A SYSTEM FOR IMPROVED PRODUCTION TITERS IN FERMENTATIONS

Abstract

The invention provides a genetically modified micro-organism for intracellular biosynthesis of a cellular metabolite, comprising a synthetic error correction system having a penalty gene, whose expression leads to arrested growth or cell death (e.g. a toxin gene) in combination with a survival gene, whose expression provides an antidote that restores cell viability and normal growth (e.g. a cognate antitoxin gene). Alternatively, the system has a survival gene, alone, whose expression is essential for growth (i.e. essential gene). The synthetic error correction system further comprises a biosensor, whose function is to induce expression of the survival gene which leads to cell growth, only, when the cell produces a pre-defined level of a given metabolite. The invention further encompasses: a method for producing the genetically modified micro-organism; a method for producing a cellular metabolite with the genetically modified micro-organism; and use of the genetically modified micro-organism for producing a cellular metabolite.

Description

TECHNICAL FIELD OF THE INVENTION

[0001] The invention provides a genetically modified micro-organism for intracellular biosynthesis of a cellular metabolite, comprising a synthetic error correction system having a penalty gene, whose expression can either lead to arrested growth or cell death (e.g. a toxin gene) in combination with a survival gene, whose expression provides an antidote that can restore cell viability and normal growth (e.g. a cognate antitoxin gene). Alternatively, the system has a survival gene, alone, whose expression is essential for growth (i.e. essential gene). Additionally, the synthetic error correction system has a biosensor, whose function is to induce expression of the survival gene leading to cell growth, only, when the cell produces a pre-defined level of a given metabolite. The invention further encompasses: a method for producing the genetically modified micro-organism; a method for producing a cellular metabolite with the genetically modified micro-organism; and use of the genetically modified micro-organism for producing a cellular metabolite.

BACKGROUND OF THE INVENTION

[0002] An increasing share of the world's chemical production relies on microorganisms or mammalian cells that are genetically engineered to function as cell factories, and tailor-made for the biosynthesis of a given molecule. Production processes, employing these cell-factories, are typically initiated from a starter culture of a small number of cells of a production organism, which go through a phase of growth and expansion of cell numbers in large fermentation tanks (up to 30,000 L volume). In some setups, production of a given molecule proceeds both during the growth phase and during a subsequent period (batch and fed-batch cultures). Alternatively, in order not to waste resources associated with cleaning and growing up a new batch, production is continuous. A chemostat fermentor allows a production organism to be grown in a fermentation broth that is constantly diluted, thus tapping product and cells from the culture, while replenishing with fresh nutrient medium. On an industrial scale, such production processes may continue operation for 1-2 months before starting a new culture in a clean tank. The fermentation processes and equipment used in this industry are very similar, both for the production of a wide range of commodity small molecules and for therapeutic proteins, and consequently these processes are subject to similar problems.

[0003] In particular, the appearance of non-producing cells (unable to produce the product molecule) is commonly observed, especially when the production run is for extended periods of time (chemostat). Such non-producing cells within an industrial fermentation are highly undesirable, as they consume nutrients, oxygen and space. Furthermore, non-producing cells can have a selective advantage over producing cells, and may as such grow faster. In a growing cell culture, such improvements in fitness can lead to significant out-competition of the producing cells over time. This drift from the optimal production state is an eventual reason for discarding the fermentation broth and spending resources on cleaning, sterilization, not to mention nutrients, to replenish the fermentation tank with new, producing organisms. Such non-producing cells originate from genetic mutations that arise in the cells of an original producing organism undergoing many growth divisions.

[0004] Since the occurrence of genetic mutations in cells of a production organism that lead to a loss of product formation by the cells during a production run, cannot be avoided, there is a need for methods for eliminating or slowing the growth of non-producing cells in the production. Preferably, such methods of elimination are sufficiently effective that they prevent the observed drift from the production state, and thereby prolong the life-time of an industrial fermentation.

[0005] If the system employs a penalty gene alone (and no antidote), then an "OFF" type sensor is required. If the system employs a survival gene alone (e.g. essential genes) a metabolite-linked "ON" type sensor is needed as in the first scenario.

SUMMARY OF THE INVENTION

[0006] The invention provides a genetically modified microbial cell for intracellular biosynthesis of a cellular metabolite comprising:

[0007] a) a first nucleic acid molecule wherein the transcription and/or translation of said molecule yields a biosensor capable of binding said cellular metabolite to form a complex; and any one selected from the group consisting of ((b), (c) and (d)):

[0008] b) a second nucleic acid molecule comprising a coding sequence encoding a first protein required for cell growth and/or survival, wherein the second nucleic acid molecule is operably linked to a first promoter; wherein expression of said first protein encoded by said second nucleic acid molecule is induced when said biosensor and said cellular metabolite form a complex;

[0009] c) a second nucleic acid molecule comprising a coding sequence encoding a first protein required for cell growth and/or survival, wherein the second nucleic acid molecule is operably linked to a first promoter; and

[0010] a third nucleic acid molecule encoding a second protein that is toxic for cell growth and/or survival, wherein said third nucleic acid molecule comprises a coding sequence operably linked to a second constitutive promoter; wherein expression of said first protein encoded by said second nucleic acid molecule is induced when said biosensor and said cellular metabolite form a complex; and

[0011] d) a second nucleic acid molecule encoding a protein that is toxic for cell growth and/or survival, wherein said second nucleic acid molecule comprises a coding sequence operably linked to a promoter; wherein expression of said protein is prevented when said biosensor and said cellular metabolite form a complex;

[0012] whereby arrest of growth and/or death of said cell due to an absence of complex formation does not depend on externally supplied growth inhibitor or growth retardant.

[0013] According to one embodiment of the genetically modified microbial cell of invention comprising the features of (a) and (b), the first promoter is an inducible promoter, and the first protein (encoded by the second nucleic acid molecule) is essential for growth of the cell.

[0014] According to a further embodiment, the genetically modified microbial cell of the invention comprises the features of (a) and (c), wherein:

said first promoter linked to said second nucleic acid molecule is inducible, and said second protein (encoded by the third nucleic acid molecule) is a toxin; and said first protein (encoded by the second nucleic acid molecule) is an anti-toxin protein cognate to said toxin protein; and said biosensor is a transcription factor capable of binding to said metabolite to form a complex, and wherein said complex is capable of binding to said inducible promoter so as to induce expression of the first protein.

[0015] According to a further embodiment, the genetically modified microbial cell of the invention comprises the features of (a) and (c), wherein:

said second protein (encoded by the third nucleic acid molecule) is a toxin; and wherein: said first protein (encoded by the second nucleic acid molecule) is an anti-toxin cognate to said toxin; said first nucleic acid molecule is operably linked to said second nucleic acid molecule upstream to its coding sequence and is operably linked downstream of the first promoter, and wherein the first promoter is a constitutive promoter, and said biosensor obtained on transcription of said first nucleic acid molecule is a riboswitch capable of binding to said metabolite to form a complex.

[0016] According to a further embodiment, the genetically modified microbial cell of the invention comprises the features of (a) and (d), wherein:

said protein (encoded by the second nucleic acid molecule) is a toxin; and said promoter is inducible; and and said biosensor is a transcription factor capable of binding to said metabolite to form a complex, and wherein said complex is capable of binding to said inducible promoter so as to induce expression of the protein.

[0017] According to a further embodiment, the genetically modified microbial cell of the invention comprises the features of (a) and (d), wherein:

said protein (encoded by the nucleic acid molecule) is a toxin; said first nucleic acid molecule is operably linked to said second nucleic acid molecule upstream of its coding sequence and is operably linked downstream of the first promoter, and wherein the first promoter is a constitutive promoter, and wherein said biosensor obtained on transcription of said first nucleic acid molecule is a riboswitch capable of binding to said metabolite to form a complex.

[0018] According to a further embodiment of the genetically modified microbial cell of the invention, the cellular metabolite is selected from the groups consisting of: isoprenoid(s), vitamin(s), carboxylic acid(s), amino acid(s), fatty acid(s), alcohol(s), and polyketide(s).

[0019] The invention further provides a method of genetically modifying a microbial cell for the biosynthesis of a metabolite comprising the steps of introducing into the cell:

a nucleic acid molecule encoding a toxin operably linked to a constitutive promoter; a nucleic acid molecule encoding an anti-toxin cognate to the toxin, wherein the molecule is linked to an inducible promoter; and a nucleic acid molecule wherein the transcription and/or translation of said molecule yields a biosensor capable of binding to the metabolite; wherein expression of said antitoxin is induced when said biosensor and said cellular metabolite form a complex and bind to said inducible promoter; and wherein arrest of growth and/or death of said cell due to an absence of complex formation does not depend on externally supplied growth inhibitor or growth retardant.

[0020] The invention further provides a method for producing a biosynthetic metabolite comprising the steps of:

providing a genetically modified microbial cell according to any one embodiment of the invention, introducing the genetically modified microbial cell into a cultivation medium comprising a substrate for production of said metabolite, and optionally cultured in a culture vessel by continuous culture for a period of at least 24 cell multiplications; and then recovering metabolite produced by said culture, wherein a lack of metabolite production in said genetically modified microbial cell or progeny cell thereof attenuates multiplication of said cell as compared to a non-genetically modified parent cell from which said modified microbial cell was derived.

[0021] The invention further includes the use of a genetically modified microbial cell according to any one embodiment of the invention, for producing a biosynthetic metabolite, wherein a lack of metabolite production in said genetically modified microbial cell or progeny cell thereof attenuates multiplication of said cell as compared to the metabolite producing genetically modified microbial cell.

DESCRIPTION OF THE INVENTION

Figures

[0022] FIG. 1. Cartoon of the general concept of synthetic error-correction.

[0023] FIG. 2. Cartoon of L-arabinose-addiction regulated TA-system Dual expression TA gene construct comprising the toxin gene yoeB whose expression is regulated by the p.sub.Lac promoter; and the antitoxin gene yefM whose expression is regulated by the p.sub.BAD promoter, and the araC L-arabinose sensor gene.

[0024] FIG. 3. Growth dependency through the yoeB-yefM system on induction of respectively toxin and antitoxin, as measured by cell density (OD.sub.600 nm) as a function of time (hr).

[0025] A) Cell growth in the absence of an inducer (L-arabinose) of antitoxin expression, and with a supply of IPTG (I), a toxin expression inducer, at a range of concentrations rising from 0, 0.005, 0.025, 0.1, 0.5, 1.5 mM IPTG, (corresponding to 01 to 51, respectively).

[0026] B) Cell growth in the presence of an inducer (0.1% L-arabinose) of antitoxin expression, and with a supply of IPTG (I), a toxin expression inducer, at a range of concentrations rising from 0, 0.005, 0.025, 0.1, 0.5, 1.5 mM IPTG, (corresponding to 01 to 51, respectively). Growth curves are averages (n=3).

[0027] FIG. 4. Cartoon of mevalonate addiction regulated TA-system Dual expression TA gene construct comprising the toxin gene yoeB whose expression is regulated by the p.sub.Lac promoter; and the antitoxin gene yefM, whose expression is regulated by the p.sub.BAD promoter, and the mutated araCmev mevalonate sensor gene with 4 point mutations.

[0028] FIG. 5. Cartoon of the operon encoding two mevalonate biosynthesis pathways from acetyl-CoA:

A) pMevTC operon comprising E. coli atoB encoding an acetyl-CoA acetyltransferase; Saccharomyces cerevisiae ERG13 encoding an HMG-CoA synthase, and tHMGR, a truncated version of Saccharomyces cerevisiae HMGR, encoding an HMG-CoA reductase; B) pMEV7C operon comprising E. coli atoB; a Lactococcus lactis gene mvaS encoding an HMG-CoA synthase, and a Lactococcus lactis mvaE gene encoding a HMG-CoA reductase; and C) mevalonate biosynthetic pathway: the atoB encoded acetoacetyl-CoA thiolase catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA; the ERG13/mvaS encoded HMG-CoA synthase, which creates 3-hydroxy-methylglutayl-CoA (HMG-CoA) by a condensation reaction between acetoacetyl-CoA and another molecule of acetyl-CoA; and tHMGR/mvaE encoded HMG-CoA reductase which converts HMG-CoA to mevalonate. FIG. 6. Cancelling the fitness advantage of non-producing cells. A) Cell growth over time (measured by cell density at OD.sub.600 nm) of E. coli at 30.degree. C. in 2.times.YT medium, comprising the plasmid (pMEV7C) for `high` level mevalonate synthesis and the control plasmid (pMevT5c) for no mevalonate synthesis. B) Growth curves of strains harbouring the same pathway plasmids as well as the mevalonate-TA correction system.

[0029] FIG. 7. Enhancing mevalonate production in fermentation populations using the TA system to eliminate non-producing cells. Histogram showing mevalonate (MVA) levels produced by mixture cultures with the indicated percentage (x-axis) of producing cells among non-producing cells. Producing cells comprised the mevalonate production plasmid (pMEV7C) while non-producing cells comprised the control plasmid (pMevT5c) with the inactivated production pathway. The cells were tested with/without the plasmid expressing the mevalonate-TA system for "correction" (pBAM-TA5).

[0030] FIG. 8. Cartoon showing a cloning and strain construction strategy for addiction through essential genes. The metabolite-responsive promoter is introduced in the genome of the host strain, replacing the native promoter of an essential gene (optionally operon). Upstream and downstream homologous regions (HR) guide replacement of native DNA with the responsive promoter and a kanamycin resistance gene for selection of genomic DNA insertion. Using a variable ribosome binding site (RBS) with redundant nucleotide, a wider range of responses can be screened in order to identify a host strain, which has become addiction an internally produced metabolite.

[0031] FIG. 9. Cell growth controlled by the L-arabinose-addiction regulated essential gene system. Cell growth over time (measured by cell density at OD.sub.600 nm) of E. coli comprising the essential gene operon folP/glmM driven by the p.sub.BAD promoter were cultivated at 37.degree. C. in 2.times.YT growth medium supplemented with 0% or 0.25% L-arabinose. Growth curves are averages (n=3). Error bars denote standard error.

[0032] FIG. 10. Growth of E. coli strains addicted to internal production of mevalonate. Cell growth over time (measured by cell density at OD.sub.600 nm) of E. coli strains engineered to be mevalonate-addicted through the essential gene operon folP/glmM, comprising inoculums of the 100% mevalonate producing strain (e3.9) and inoculum mixtures with the given percentages of the mevalonate producing strain (e3.9) and a mevalonate pathway non-producing strain (e3.8). Growth curves are averages (n=3). Error bars denote standard error.

[0033] FIG. 11. Fitness cost to non-addicted control cells of supplemented triacetic acid lactone (TAL). Growth of E. coli control strain e3.16CON, measured as increase in cell density (OD.sub.630 nm) over time, in 2.times.YT medium (with kanamycin and spectinomycin) supplemented with respectively 0, 2 and 20 mM triacetic acid lactone at 37 deg. C. in 200 .mu.L microtiter plates with continuous shaking. All wells were inoculated with the same number of cells. Error bars depict the standard error of the mean (n=3).

[0034] FIG. 12: Triacetic acid lactone-addicted growth of E. coli strain e3.16#5 in 2.times.YT medium (with kanamycin and spectinomycin) supplemented with respectively 0, 2 and 20 mM triacetic acid lactone (TAL), measured as increase in cell density (OD.sub.630 nm) over time. Cells of the strain were grown at 37 deg. C. in 200 .mu.L microtiter plates with continuous shaking. All wells were inoculated with the same number of cells. Error bars depict the standard error of the mean (n=3).

[0035] FIG. 13: Fitness cost to non-addicted control cells of supplemented salicylic acid. Growth of E. coli e3.18CON, measured as increase in cell density (OD.sub.630 nm) over time, in 2.times.YT medium (with kanamycin and spectinomycin) supplemented with respectively 0 or 5 mM salicylic acid and grown at 37 deg. C. in 200 .mu.L microtiter plates with continuous shaking. All wells were inoculated with the same number of cells. Error bars depict the standard error of the mean (n=3).

[0036] FIG. 14: The salicylic acid-addicted growth of E. coli e3.18#1, measured as increase in cell density (OD.sub.630 nm) over time, cultured in 2.times.YT medium (with kanamycin and spectinomycin) supplemented with respectively 0 or 5 mM salicylic acid and grown at 37 deg. C. in 200 .mu.L microtiter plates with continuous shaking. All wells were inoculated with the same number of cells.

[0037] Error bars depict the standard error of the mean (n=3).

[0038] FIG. 15: Fitness cost to control cells of supplemented benzoic acid. Growth of E. coli e3.22CON, measured as increase in cell density (OD.sub.630 nm) over time, in 2.times.YT medium (with kanamycin and spectinomycin) supplemented with respectively 0 or 5 mM benzoic acid and grown at 37 deg. C. in 200 .mu.L microtiter plates with continuous shaking. All wells were inoculated with the same number of cells. Error bars depict the standard error of the mean (n=3).

[0039] FIG. 16: Benzoic acid-addicted growth of E. coli e3.22#4, measured as increase in cell density (OD.sub.630 nm) over time, in 2.times.YT medium (with kanamycin and spectinomycin) supplemented with respectively 0 or 5 mM benzoic acid and grown at 37 deg. C. in 200 .mu.L microtiter plates with continuous shaking. All wells were inoculated with the same number of cells. Error bars depict the standard error of the mean (n=3).

[0040] FIG. 17. Cartoon of a DNA construct comprising a TPP-addiction riboswitch-regulated TA system.

[0041] FIG. 18. Cell growth controlled by the thiamine pyrophosphate (TPP)-responsive riboswitch linked to a TA system. Cell density following 15 hours of incubation at 37.degree. C. (measured by cell density at OD.sub.600 nm) of E. coli comprising TPP-responsive riboswitch controlling the translational rate of the antitoxin, YefM, and a constitutively expressed toxin, YoeB (0 .mu.M IPTG) or induced toxin by LacI with IPTG inducer (5, 50 and 500 .mu.M IPTG). Cell density measured in presence and absence of 500 .mu.M TPP added to the growth medium.

[0042] FIG. 19. Improvement with metabolite-addiction system following long-term culture. Concentration of mevalonate (MVA) accumulated in E. coli XL1 featuring the same MVA pathway plasmid following a total of 55 cell generations. Strain e3.9 unlike pe1 further features a chromosomal change in the promoter of an essential gene operon rendering its expression dependent on the product, MVA. Error bars denote standard deviation of biological replicates (n=3).

ABBREVIATIONS AND TERMS

[0043] gi number: (genInfo identifier) is a unique integer which identifies a particular sequence, independent of the database source, which is assigned by NCBI to all sequences processed into Entrez, including nucleotide sequences from DDBJ/EMBL/GenBank, protein sequences from SWISS-PROT, PIR and many others

[0044] Amino acid sequence identity: The term "sequence identity" as used herein, indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length. The two sequences to be compared must be aligned to give a best possible fit, by means of the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as ((Nref-Ndif)100)/(Nref), wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson W. R and D. J. Lipman (1988)) (www.ncbi.nlm.nih.gov/cgi-bin/BLAST). In one embodiment of the invention, alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al 1994, available at http://www2.ebi.ac.uk/clustalw/.

[0045] Preferably, the numbers of substitutions, insertions, additions or deletions of one or more amino acid residues in the polypeptide as compared to its comparator polypeptide is limited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deletions. Preferably the substitutions are conservative amino acid substitutions: limited to exchanges within members of group 1: Glycine, Alanine, Valine, Leucine, Isoleucine; group 2: Serine, Cysteine, Selenocysteine, Threonine, Methionine; group 3: Proline; group 4: Phenylalanine, Tyrosine, Tryptophan; Group 5: Aspartate, Glutamate, Asparagine, Glutamine.

[0046] Native gene: endogenous gene in a microbial cell genome, homologous to host micro-organism.

[0047] Biosensor: Suitable biosensor effectors (small molecules or metabolites detectable by a sensor) and their sensors can be found in the RegPrecise Collection of Manually Curated Inferences of Regulons in Prokaryotic Genomes, available at: http://regprecise.lbl.gov/RegPrecise/collections_effector.jsp

[0048] Cell multiplication: process whereby cells multiply in number, as one cell divides into two cells, these two cells both divides to become four cells, four cells become eight, eight cells become sixteen etc. Each round of cell multiplication, by cell division, yields a new cell generation.

[0049] Cognate: in the context of Toxin-Antitoxin systems; an antitoxin protein interacts with its cognate toxin to neutralise the activity of the toxin.

[0050] Fitness cost: reduction in growth rate of a microbial cell e.g. due to producing a small molecule or metabolite (including a protein), measured relative to the growth rate of a reference microbial cell where production is inactivated. Fitness cost of a metabolic pathway can also be observed as an increase in growth lag phase, again relative to a reference cell where this pathway is inactivated.

[0051] Growth inhibitor/retardant of external origin: in the context of the invention, a growth inhibitor/retardant is a component of external origin that can inhibit or retard the growth of a micro-organism (such as a toxin or antibiotic), that is not produced by the micro-organism itself, but instead is supplied to the micro-organism either by addition to or its presence in the growth medium or environment in which the micro-organism is cultured.

[0052] Operably linked: a gene (nucleic acid molecule comprising a coding sequence) is operably linked to a promoter when its transcription is under the control of the promoter and where transcription results in a transcript whose subsequent translation yields the product encoded by the gene. Similarly a first nucleic acid molecule encoding a riboswitch may be operably linked to a second nucleic acid molecule upstream of its coding sequence; and also operably linked downstream of a promoter; whereby transcription of the downstream first nucleic acid molecule comprising the riboswitch linked to the second nucleic is under the control of the promoter and results in a transcript; and whereby translation of the transcript so produced (comprising the riboswitch and coding sequence) is under the control of the riboswitch.

[0053] Toxic: in the context of the invention a protein is defined as toxic if arrests or limits cell growth and/or prevents cell survival; e.g. a protein toxin.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention aims to prolong the productive life-time of an industrial fermentation by preventing the observed drift of a population of cells in an industrial fermentation from a productive state to a non-productive state. This drift typically arises as a result of spontaneous genetic mutations in cells of the production organism during continuous growth, and where a competitive advantage of a non-producing mutant favors its proliferation.

I: A Genetically Modified Microbial Cell Comprising a Synthetic Error Correction System Controlled by a Metabolite Biosensor

[0055] The invention is based on the general concept of a synthetic error correction system which is illustrated in FIG. 1. This system uses either a penalty gene, whose expression can either lead to arrested growth or cell death (e.g. a toxin gene) in combination with a survival gene, whose expression provides an antidote that can restore cell viability and normal growth (e.g. a cognate antitoxin gene). Alternatively, the system uses a survival gene, alone, whose expression is essential for growth (i.e. essential gene).

[0056] The second key component of the system is a biosensor, whose function is to induce an appropriate gene regulation response leading to cell growth only when the cell produces a pre-defined level of a given metabolite. Cell survival and growth is said to be "addicted" to the presence of its metabolite i.e. to its addiction molecule. The functional properties of the biosensor depend on the type of gene regulation response required. If the system employs a penalty gene (such as a toxin gene) in combination with a survival gene (a cognate anti-toxin gene), then cell survival requires an "ON" type sensor linked to the expression of the survival gene. If the system employs a penalty gene alone (and no antidote), then an "OFF" type sensor is required. If the system employs a survival gene alone (e.g. essential genes) a metabolite-linked "ON" type sensor is needed as in the first scenario.

[0057] Accordingly, the fate of a cell, namely its survival and growth versus its arrested growth and eventual death, when employing this system, is determined by its continued production of a given metabolite, which is the product, or a close biosynthesis intermediate of the product of the industrial fermentation. A key feature of the synthetic error correction system in cells of the invention, is that the penalty executed by means of the error correction system in non-producing cells is realized by the expression of the penalty gene(s) and/or the failure to express the survival gene(s) (i.e. the expression of these genes and the cellular products thereof is both necessary and sufficient for achieving error correction). Accordingly, the arrest of growth or death of non-producing cells of the invention does not require the presence or addition of externally supplied compounds (e.g. toxins or antibiotics) for the execution of the penalty. This is an advantageous feature of the present invention, since the use of antibiotics or other growth retardants in the cultivation medium during industrial scale microbial fermentation would compromise the economics, biosafety and stability of production. The invention provides a genetically modified microbial cell for use in the intracellular biosynthesis of a cellular metabolite, comprising the above described synthetic error correction system. The genetically modified microbial cell comprises at least: a first nucleic acid molecule wherein the transcription and/or translation of said molecule yields a biosensor capable of binding the cellular metabolite to form a complex; and a second nucleic acid molecule comprising a coding sequence operably linked to a promoter, and encoding a protein required for cell growth and/or survival; and optionally a third nucleic acid molecule comprising a coding sequence operably linked to a promoter, and encoding a protein that inhibits the growth and/or survival of the cell. The expression of the protein encoded by the second nucleic acid molecule is induced when the biosensor and the cellular metabolite form a complex. Various embodiments of the genetically modified microbial cell of the invention are described below:

II: A Genetically Modified Microbial Cell Comprising a Toxin-Antitoxin System Controlled by a Metabolite Biosensor

[0058] According to a first embodiment, the genetically modified microbial cell, which is for use in the intracellular biosynthesis of a given cellular metabolite, is a cell comprising a toxin-antitoxin system controlled by a metabolite biosensor which has at least the following features:

1. a first nucleic acid molecule encoding a transcription factor that functions as a biosensor and is capable of binding to the cellular metabolite produced by the cell to form a complex. The complex functions as an "activated transcription complex" in that it is able to interact with a gene promoter and induce expression of its cognate coding sequence; and 2. a second nucleic acid molecule which is operably linked to an inducible promoter and comprises a coding sequence encoding an anti-toxin protein that is cognate to and binds to the toxin, whereby the anti-toxin acts as an antidote to the toxin and permits the survival and growth of the cell; and 3. a third nucleic acid molecule encoding a toxin protein wherein the nucleic acid molecule comprises a coding sequence that is operably linked to a constitutive promoter; wherein expression of the antitoxin encoded by the second nucleic acid molecule is induced when the transcription factor and the cellular metabolite form a complex. As stated above, this complex, which functions as an "activated transcription complex" is capable of binding to the inducible promoter of the second nucleic acid molecule so as to induce expression of the antitoxin.

[0059] The Toxin/Antitoxin (TA) system, as used herein, is a two-component system whose features are used to slow the growth, or eliminate, non-producing micro-organisms that arise in a proliferating cell population during production of a metabolite e.g. during fermentation. The general concept of TA systems is given by the name: one component is a toxin molecule which affects a critical function in a cell, and the second component is an antitoxin that, upon expression in the cell, can cancel the effect of the toxin. The toxin usually causes growth arrest or cell death by impairing functions such as transcription, translation, cell division (replication and cytoskeleton formation), or membrane stability.

[0060] Examples of suitable TA systems include the type II TA pair, yefM-yoeB, from E. coli. The YoeB protein is a toxin that functions as an mRNA interferase, and binds to the 50S subunit of the ribosome to block translation initiation. The YoeB protein also has endoribonuclease activity without association to the ribosome. The antitoxin, YefM, forms a dimer which binds a single YoeB molecule to form a stable complex, which inactivates the mRNA-degrading action of YoeB. The YefM antitoxin is very sensitive to degradation by the Lon protease, whereby YoeB is then released and causes a growth arrest. Since the antitoxin has a short half-life, the absence of cellular metabolite required for continued expression of the antitoxin rapidly leads to release of the toxin and subsequent growth arrest.

[0061] The amino acid sequence of a functional YefM anti-toxin encoded by the coding sequence of the second nucleic acid molecule, has at least 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99, or 100% sequence identity to SEQ ID No: 2.

[0062] The amino acid sequence of a functional YoeB toxin encoded by the coding sequence of the third nucleic acid molecule, has at least 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99, or 100% sequence identity to SEQ ID No: 3.

[0063] Alternative suitable TA pairs include mazF-mazE (SEQ ID No. 36 and 38; encoded by SEQ ID No: 35 and 37 respectively), yafO-yafN (SEQ ID No. 40 and 42; encoded by SEQ ID No: 39 and 41 respectively) and relE-relB (SEQ ID No. 44 and 46; encoded by SEQ ID No: 43 and 45 respectively).

[0064] Use of alternative, suitable TA pairs when using the pBAD-TA5 vector requires that the DNA sequences encoding yoeB and yefM in pBAD-TA5 are replaced with the respective toxin and antitoxin encoding sequence above.

[0065] The third nucleic acid molecule comprises a constitutive promoter, operably linked to a coding sequence encoding a toxin protein that drives expression of the toxin protein. A suitable promoter is a constitutive promoter, whereby toxin protein is expressed continuously within the cell, for example J23100 having nucleotide sequence SEQ ID No: 48.

[0066] The second nucleic acid molecule comprises a coding sequence encoding an anti-toxin protein, operably linked to an inducible promoter that drives expression of the anti-toxin protein. A suitable inducible promoter is one that is activated and induces expression of the cognate coding sequence encoding the antitoxin protein, when the biosensor (a transcription factor) and the cellular metabolite form a complex. More specifically, transcription (and expression of the anti-toxin protein) may be induced on binding of this complex to this inducible promoter.

[0067] By way of example only, a suitable inducible promoter includes the p.sub.BAD (SEQ ID No: 66), that is inducible by the transcription factor biosensors that bind to the metabolites L-arabinose, and mevalonate, (see examples).

[0068] The first nucleic acid molecule encodes a transcription factor that is capable of binding to the cellular metabolite produced by the cell to form a complex. The complex functions as an "activated transcription complex" in that it is able to interact with a gene promoter and induce expression of its cognate coding sequence.

[0069] By way of example only, suitable transcription factors encoded by the coding sequence of the first nucleic acid molecule include AraC (SEQ ID No: 6), AraCmev (SEQ ID No: 12) and FadR (SEQ ID No: 68) that function as biosensors by binding to the metabolites L-arabinose, mevalonate and fatty acid/fatty acid acyl-CoA respectively (see examples illustrating biosensor:inducible promoter pairs for a range of metabolites).

[0070] The first nucleic acid molecule comprises a promoter operatively linked to a coding sequence encoding the transcription factor. If a eukaryotic host cell is used, the transcription factor should comprise a nuclear localization signal peptide e.g. encoding the protein sequence Pro-Lys-Lys-Lys-Arg-Lys-Val.

[0071] A suitable promoter is a constitutive promoter, whereby the biosensor (transcription factor) is expressed continuously within the cell at a level where binding of effector yields responsive gene regulation, for example selected from among the synthetic promoters listed below, for example J23100 having nucleotide sequence of SEQ ID No: 48.

TABLE-US-00001 SEQ ID Promoter Sequence (5'-) No J23119 ttgacagctagctcagtcctaggtataatgctagc 47 J23100 ttgacggctagctcagtcctaggtacagtgctagc 48 J23101 tttacagctagctcagtcctaggtattatgctagc 49 J23102 ttgacagctagctcagtcctaggtactgtgctagc 50 J23103 ctgatagctagctcagtcctagggattatgctagc 51 J23104 ttgacagctagctcagtcctaggtattgtgctagc 52 J23105 tttacggctagctcagtcctaggtactatgctagc 53 J23106 tttacggctagctcagtcctaggtatagtgctagc 54 J23107 tttacggctagctcagccctaggtattatgctagc 55 J23108 ctgacagctagctcagtcctaggtataatgctagc 56 J23109 tttacagctagctcagtcctagggactgtgctagc 57 J23110 tttacggctagctcagtcctaggtacaatgctagc 58 J23111 ttgacggctagctcagtcctaggtatagtgctagc 59 J23112 ctgatagctagctcagtcctagggattatgctagc 60 J23113 ctgatggctagctcagtcctagggattatgctagc 61 J23114 tttatggctagctcagtcctaggtacaatgctagc 62 J23115 tttatagctagctcagcccttggtacaatgctagc 63 J23116 ttgacagctagctcagtcctagggactatgctagc 64 J23117 ttgacagctagctcagtcctagggattgtgctagc 65 J23118 ttgacggctagctcagtcctaggtattgtgctagc 89

III: A Genetically Modified Microbial Cell Comprising a Toxin-Antitoxin System Controlled by a Metabolite Riboswitch Biosensor

[0072] According to a second embodiment, the genetically modified microbial cell, which is for use in the intracellular biosynthesis of a given cellular metabolite, is a cell comprising a toxin-antitoxin system controlled by a metabolite biosensor which has at least the following features:

1. a first nucleic acid molecule, and 2. a second nucleic acid molecule comprising a coding sequence, wherein the first nucleic acid molecule is operably linked to the second nucleic acid molecule upstream if its coding sequence and operably linked downstream of a constitutive promoter; and wherein the second nucleic acid molecule comprises a coding sequence encoding an anti-toxin protein that is cognate to and binds to the toxin, whereby the anti-toxin acts as an antidote to the toxin and permits the survival and growth of the cell; and wherein the transcription product of the first nucleic acid molecule is a a riboswitch capable of binding to the cellular metabolite to form a complex; 3. a third nucleic acid molecule encoding a toxin protein wherein the nucleic acid molecule comprises a coding sequence that is operably linked to a constitutive promoter; wherein expression of the antitoxin encoded by the second nucleic acid molecule is induced when the riboswitch and the cellular metabolite form a complex.

[0073] The riboswitch system, as used herein, is a method of regulating expression of the component genes of the synthetic error correction system in the genetically modified micro-organisms of the invention. The regulation takes place at the translational level, and is mediated by mRNA structures which can be formed upstream or downstream of the coding region in the 3'- or 5'-untranslated region (UTR). These riboswitches are RNA structures, which are capable of binding effectors (e.g. small-molecules or metabolites) and modulate transcription or translation of a gene in cis. A riboswitch is composed of two separate domains: an aptamer domain responsible for ligand recognition and binding, and an expression system. These two typically overlap, and the overlap is known as a switching sequence, since it will base pair with either domain depending on the state of the riboswitch. Riboswitches can also modulate gene translation by forming a structure prone to degradation by RNases. Riboswitches can be both ON and OFF switches upon ligand binding, and transcriptional control can be carried out by the formation of terminators and anti-terminators (or anti-anti-terminators) in response to molecule recognition. Translational regulation is also carried out by affecting the availability of the ribosome binding site. The riboswitch two-dimensional structure will either sequester or expose the ribosome binding site upon binding of the small molecule, resulting in the existence of both ON switches (Ribosome binding site exposed upon molecule binding), and in the opposite case, OFF switches, for example by forming a `road-block` preventing progress of the already bound ribosomal machinery.

[0074] By way of example, the nucleotide sequence of a first nucleic acid molecule that is transcribed into a suitable OFF riboswitch is selected from the group consisting of: btuB leader (adenosylcobalamin-responsive) [SEQ ID No: 69]; tc3 (tetracycline-responsive) [SEQ ID No: 70]; and ThiMwt (TPP-responsive) [SEQ ID No: 71].

An ON riboswitch, ThiMN15#19, is exemplified in Example 3.

IV: A Genetically Modified Microbial Cell Comprising a Toxin Gene Controlled by a Metabolite OFF-Type Biosensor

[0075] According to a third embodiment, the genetically modified microbial cell, which is for use in the intracellular biosynthesis of a given cellular metabolite, is a cell comprising a toxin system controlled by a metabolite biosensor which has at least the following features:

1. a first nucleic acid molecule wherein the transcription and/or translation of said molecule yields a biosensor capable of binding said cellular metabolite to form a complex, and 2. a second nucleic acid molecule which is operably linked to a constitutive promoter and where the second nucleic acid molecule comprises a coding sequence encoding a toxin protein that prevents the survival and/or growth of the cell; and wherein the complex functions to block the transcription and/or translation of the second nucleic acid molecule.

[0076] When the first nucleic acid encodes a transcription factor, the complex that is formed on its binding to the cellular metabolite is an "activated transcription complex" that is capable of interacting with the promoter of the second nucleic acid molecule so as to block expression of the toxin.

[0077] Alternatively, first nucleic acid molecule is operably linked to the second nucleic acid molecule upstream of its coding sequence of and operably linked downstream of the constitutive promoter, said biosensor obtained on transcription of said first nucleic acid molecule is a riboswitch capable of binding to said metabolite to form a complex, and thereby blocking expression of the toxin.

[0078] A suitable "OFF" transcription factor for this third embodiment includes XylR (repressor) biosensor (xylose-responsive) [SEQ ID No: 72] and its cognate responsive promoter [SEQ ID No: 73]); and a suitable "OFF" riboswitch for this variant of the third embodiment includes ThiMwt (TPP-responsive, SEQ ID No: 74)

V: A Genetically Modified Microbial Cell Comprising an Essential Gene Controlled by a Metabolite Biosensor

[0079] According to a fourth embodiment, the genetically modified microbial cell, which is for use in the intracellular biosynthesis of a given cellular metabolite, is a cell comprising an essential gene whose expression is controlled by a metabolite biosensor and which has at least the following features:

1. a first nucleic acid molecule encoding a transcription factor that functions as a biosensor and is capable of binding to the cellular metabolite produced by the cell to form a complex. The complex functions as an "activated transcription complex" in that it is able to interact with a gene promoter and induce expression of its cognate coding sequence; and 2. a second nucleic acid molecule (also known as an essential gene), which is operably linked to an inducible promoter, and comprises a coding sequence encoding an essential protein, and wherein expression of this essential protein permits the survival and growth of the cell; and wherein expression of the essential protein encoded by the second nucleic acid molecule is induced when the transcription factor and the cellular metabolite form a complex. As stated above, this complex, which functions as an "activated transcription complex" is capable of binding to the inducible promoter of the second nucleic acid molecule so as to induce expression of the essential protein. Examples of biosensors, encoded by the first nucleic acid molecule, and inducible promoters in the second nucleic acid molecule that control expression of the essential protein, are described in section II.

[0080] Essential genes, as used herein, provide an alternative synthetic error correction system, whereby the expression of a single gene product is used to control the fate of the genetically modified micro-organism of the invention. As the name reveals, the products of essential genes include those found necessary for cell growth under a defined set of conditions, as well as genes that become essential for growth of the genetically modified micro-organism of the invention. These conditions include the criteria that most of the necessary components for growth are present, alongside a temperature allowing for optimal growth rates. Characteristic of E. coli essential genes is that it is not possible to create viable cells of E. coli with knock-outs of these genes under the defined set of conditions.

[0081] By way of example only, the following six essential genes are suitable for use as this alternative synthetic error correction system. A characteristic of the following six essential genes is that their over-expression is not lethal for the cell:

murZ [SEQ ID No. 75], encodes an enzyme in the first committed step of peptidoglycan biosynthesis (also known as MurA) [SEQ ID No. 76]; mraY [SEQ ID No. 77], encodes a membrane-bound translocase [SEQ ID No. 78] also termed a UDP-MurNAc-pentapeptide phosphotransferase, located at the inner membrane of the ER which, alongside the gene product of murG, facilitates lipid II synthesis; glmM [SEQ ID No. 79], encodes a phosphoglucosamine mutase [SEQ ID No. 80]; The g/mM gene in E. coli encodes a phosphoglucosamine mutase; which catalyzes the isomeric conversion between glucosamine-6-phosphate and glucosamine-1-phosphate. This reaction is one of the first in the biosynthetic pathway leading to the metabolite precursor UDP-Nacetylglucosamine (UDPGlcNAc). This compound forms a branch-point for pathways leading to both peptidoglycan and lipopolysaccaride synthesis, which are both essential cell wall constituents; murI [SEQ ID No. 81], encodes a glutamase racemase [SEQ ID No. 82], required for synthesis of D-glutamic acid--another essential building block of peptidoglycan, also known as btuB; ribA [SEQ ID No. 83], encodes a GTP cyclohydrolase II [SEQ ID No. 84], which catalyses the first committed step in riboflavin biosynthesis; adk [SEQ ID No. 85], encodes an adenylate kinase [SEQ ID No. 86], which is an essential part of the nucleotide metabolism and catalyzes phosphorylation of AMP to ADP and dAMP to dATP (and nucleoside diphosphates to their correspond triphosphates).

[0082] In one embodiment the essential gene is g/mM, which is present in a two-gene operon with another essential gene: folP. The folP gene functions as the essential gene because it is the first gene in this operon, but as seen in the examples, the presence of more than one essential gene in the synthetic error correction system can be used to control the fate of the genetically modified micro-organism of the invention. The folP gene [SEQ ID No: 87], encodes dihydropteroate synthase [SEQ ID No. 88], which is part of the enzymatic pathway leading to production of tetrahydrofolate (vitamin B9). This compound is essential to normal cell growth, as folic acid cofactors are necessary for production of purines, methionine, thymidine, lysine and pantothenic acid.

VI a Cellular Metabolite Produced by a Genetically Modified Micro-Organism of the Invention

[0083] A cellular metabolite produced by intracellular biosynthesis by the genetically modified micro-organism of the invention may range from a small molecule up to larger products, such as proteins.

[0084] A small molecule, by way of example only, is mevalonate, which is a precursor to a diverse group of compounds termed isoprenoids. Isoprenoids cover a group of chemicals with a diverse range of functions, structures, and applications. With over 50.000 known compounds, isoprenoid functions include flavors and perfumes, hormones, mediators of membrane fluidity, and pharmaceuticals. Plants have especially been found to be an incredible reservoir of these diverse secondary metabolites. The existing method of obtaining these interesting molecules (e.g. by plant extraction) is however inefficient. Microbial production of isoprenoids represents a green and feasible alternative to obtaining these compounds.

[0085] Further small molecules, may include fatty acid ethyl esters and other biodiesel molecules, which represent another branch of biochemicals that can be produced in microbial cells, ultimately converting glucose or other carbon sources into combustible fuels. Polyunsaturated fatty acids, and many other metabolic products, such as amino acids and organic acids, are already existing or potential fermentation products that can be produced by the microbial cells of the invention and for which synthetic error correction system of the invention can be used, making use of known or adapted transcription or translation regulatory elements. For example, natural riboswitches have been found responsive to various vitamin B family molecules.

[0086] The present invention provides a powerful tool for enhancing the productivity of isoprenoid production by microbial cell factories, by eliminating non-producers from continuous fermentation. This is illustrated with respect to mevalonate production in the examples herein.

[0087] By way of example, a genetically modified micro-organism for production of mevalonate comprises three genes encoding a biosynthetic pathway for the production of mevalonate from acetyl-CoA, namely: the E. coli gene atoB, and the two genes HMGS (ERG13), and tHMGR from S. cerevisiae. The atoB gene encodes an acetoacetyl-CoA thiolase, which catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA. The HMGS gene encodes a HMG-CoA synthase, which forms 3-hydroxy-methylglutayl-CoA (HMG-CoA) by a condensation reaction between acetoacetyl-CoA and another molecule of acetyl-CoA. Finally, HMG-CoA is converted to mevalonate by the tHMGR gene product, which is a truncated version of an HMGR gene. The product of HMG-CoA synthase activity is toxic to the cell, and it is the accumulation of HMG-CoA which promotes growth inhibition.

[0088] There is a fitness cost related to production of mevalonate, both in the length of the lag phase and the final OD of a producing strain (FIG. 6A). Both of these traits underline the potential effect of having non-producers appear in a fermentation. As the culture is inoculated, the producing cells will have a long lag phase, leaving room for non-producers to grow and overtake the population. As mutations would most likely appear in relation to growth, this phenomenon would likely be of greater importance when transferring a growing culture into fresh medium, at which point cheater cells had already appeared in the previous cultivation. This would also apply to batch fermentations, where one or more serial pre-cultures are used, as well as for chemostat cultures. As presented in the examples, the TA-correction system is shown to be effective in reducing the relative fitness cost between a producing and non-producing strain by lowering the fitness of the non-producers.

VII Methods for Producing a Cellular Metabolite Using the Genetically Modified Micro-Organism of the Invention

[0089] According to a further embodiment, the invention provides a method for producing a biosynthetic metabolite comprising the steps of: a) providing a genetically modified microbial cell as defined above in sections I--IV, b) introducing the genetically modified microbial cell into a cultivation medium comprising a substrate for production of said metabolite, and c) recovering metabolite produced by said culture, wherein a lack of metabolite production in said genetically modified microbial cell or progeny cell thereof attenuates multiplication of said cell as compared to a non-genetically modified parent cell from which said modified microbial cell was derived.

[0090] In step b) it is contemplated that the cell in culture medium are cultivated under continuous; fed-batch or batch culture; and that the cells undergo at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 generations of cell multiplication. The period of cultivation will depend on the micro-organism cultivated; but where the micro-organism is a bacterial cell, the accumulated period of cultivation is typically at least 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 days. In the context of the present invention the term "accumulated period of cultivation" is to be understood to include the cultivation of a pre-seed culture; further cultivation after subsequent inoculation of the pre-seed culture into a larger fermenter, and optionally cultivation after subsequent inoculation of the previous culture into an even larger fermenter. Production of a metabolite using the genetically modified microbial cell of the invention wherein the production of the metabolite has a high fitness cost of production, such as .gtoreq.5%, .gtoreq.10%, .gtoreq.15%, .gtoreq.20%, and .gtoreq.25%.

VIII Micro-Organisms for the Intracellular Biosynthesis of a Cellular Metabolite

[0091] The micro-organism for the intracellular biosynthesis of a cellular metabolite according to the invention, may be a bacterium, a non-exhaustive list of suitable bacteria is given as follows: a species belonging to the genus Bacillus, a species belonging to the genus Escherichia, a species belonging to the genus Lactobacillus, a species belonging to the genus Lactococcus, a species belonging to the genus Corynebacterium, a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas; a species belonging to the genus Proprionibacterium, and a species belonging to the genus Bifidobacterium.

[0092] Alternatively, the micro-organism according to the invention may be a yeast belonging to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum; a yeast belonging to the genus Kluyveromyces, e.g. K. lactis K. marxianus var. marxianus, K. thermotolerans; a yeast belonging to the genus Candida, e.g. C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis; a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, or other yeast genera, e.g. Cryptococcus, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces or Schizosaccharomyces. Alternatively, the micro-organisms may be a filamentous fungus belonging to the genus of Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma Thermomyces, Streptomyces and Aspergillus. More specifically, the micro-organism may be Fusarium Oxysporum, A. niger, A. awamori, A. oryzae, and A. nidulans.

[0093] The preferred micro-organisms of the invention may be S. cerevisiae, E. coli, L. lactis or L. plantarum. Bacillus subtilis, B. licheniformis, Trichoderma resei, Aspergillus niger, Aspergillus oryzae, Yarrowia lypolytica, and Pichia pastoris.

IX Methods for Producing a Micro-Organism of the Invention

[0094] Integration and self-replicating vectors, suitable for cloning and introducing a first, second, third or additional nucleic acid molecules into a micro-organism for the intracellular biosynthesis of a cellular metabolite, are commercially available and known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). Cells of a micro-organism are genetically engineered by the introduction into the cells of heterologous DNA (RNA). Heterologous expression of genes encoding one or more polypeptide of the correction system in a micro-organism of the invention is demonstrated in the Examples.

[0095] A first, second, third or additional nucleic acid molecule(s) according to the invention, can be introduced into a cell or cells on plasmids or optionally integrated into the host cell genome using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc.

EXAMPLES

Example 1. TA Systems

1.1 L-Arabinose Addiction Coupled to a Type II TA System is Shown to Control Bacterial Cell Growth

[0096] The yefM-yoeB TA pair comprises two genes, where the yoeB gene encodes a toxin, and the yefM gene encodes its cognate antitoxin (FIG. 2). The YoeB toxin has two properties that serve to block cell growth: 1) acting as an mRNA interferase, and binding to the 50S subunit of the ribosome to block translation initiation; 2) acting as an endoribonuclease, and degrading mRNA independent of the ribosome. When a cognate YefM dimer is expressed, this binds a single YoeB molecule to form a stable complex and thereby prevents the growth inhibitory properties of the toxin. The half-life of the YefM antitoxin is short, since it is rapidly proteolytically degraded (Lon protease). Accordingly, a failure to express the YefM antitoxin leads to release of the YoeB toxin, which in turn arrests cell growth.

[0097] The use of the yefM-yoeB TA pair system to control the growth of bacteria based on L-arabinose addiction was demonstrated in E. coli, as follows.

[0098] The yefM-yoeB TA pair was cloned into a plasmid (pBAD-TA5: araC, p.sub.BAD-yefM, p.sub.Lac-yoeB, ampR) (Table 1) employing standard PCR cloning protocols (known in the art) and the plasmid was transformed into the host stain E. coli XL-1 with the genotype: recA1endA1 gyrA96 thi-1 hsdR17 supE44 re/A1 lac[F'proAB lacIqZM15 Tn10 (TetR)]. Electroporation recovery of transformed cells was carried out in SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose).

[0099] Transformed E. coli cultures were grown in 2.times.YT medium (10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl). Antibiotics were added to select for maintenance of plasmids in strains transformed with these, according to their respective antibiotic resistance gene. Antibiotic concentrations when added were as follows: ampicillin 100 .mu.g/mL, chloramphenicol 30 .mu.g/mL, spectinomycin 50 .mu.g/mL, kanamycin 50 .mu.g/mL.

[0100] The expression of the yoeB gene in the yefM-yoeB TA pair construct (FIG. 2) was placed under the control of the Lac promoter, which is inducible by isopropyl .beta.-D-1-thiogalactopyranoside (IPTG). Exposure of the cells to increasing concentrations of IPTG induced sufficient expression of the YoeB toxin to prevent growth, when the cells were grown in the absence of any source of L-arabinose (FIG. 3A). The expression of the yefM gene is placed under the control of the p.sub.BAD promoter, which is inducible by L-arabinose.

[0101] The regulation of the p.sub.BAD promoter by AraC is dual. In the absence of L-arabinose, the AraC protein binds to operator sites within p.sub.BAD effectively repressing transcription. Upon binding of L-arabinose, the complex changes to another DNA-binding conformation leading to activation of p.sub.BAD and induction of transcription of a cognate gene.

[0102] While cell growth was strongly inhibited by addition of .gtoreq.0.1 mM IPTG of the toxin inducer; the co-addition of 0.1% L-arabinose was sufficient to restore wild-type growth in the presence of IPTG at concentrations up to 0.5 mM (FIG. 3B).

[0103] These data demonstrate that the TA system has a wide dynamic range, with gradual response in respect of growth inhibition. Thus a small change in the level of toxin expression relative to antitoxin expression, does not give rise to a dramatic change in cell growth rates, but is still provides sufficiently strong growth control to prevent growth as well as allow normal growth at extremes in the concentration of the inducer L-arabinose.

[0104] Table 1 listing the genetic features of the L-arabinose TA addicted strain based on the E. coli XL1 parent strain.

TABLE-US-00002 TABLE 1 Plasmid/ chromosome Gene SEQ ID No: Protein SEQ ID No pBAD-TA5 araC* SEQ ID No: 1 p.sub.BAD-yefM YefM SEQ ID No: 2 p.sub.Lac-yoeB YoeB SEQ ID No: 3 ampR Beta- SEQ ID No: 4 lactamase araC SEQ ID No: 5 araC SEQ ID No: 6 *araC gene [SEQ ID No: 5] encoding araC biosensor protein [SEQ ID No: 6] is located on the complementary strand to SEQ ID No: 1 at nucleotide positions: 96-974. The p.sub.BAD promoter is located a position 1246-1318 in SEQ ID No: 1.

[0105] Table 2 listing the genetic features of the mevalonate TA addicted strain m14 based on the E. coli XL1 parent strain. The chromosomally located sequence is an excerpt of the genome sequence that fully comprises the engineered features.

TABLE-US-00003 TABLE 2 Plasmid/ chromosome Gene SEQ ID No: Protein SEQ ID No pBAM-TA5 araCmev* SEQ ID No: 7 p.sub.BAD-yefM YefM SEQ ID No: 8 p.sub.Lac-yoeB YoeB SEQ ID No: 9 ampR Beta- SEQ ID No: 10 lactamase araCmev SEQ ID No: 11 araCmev SEQ ID No: 12 chromosome yefM-FRT- SEQ ID No: 13 Neomycin SEQ ID No: 14 kanR-FRT- phospho- yoeB** transferase II *araCmev gene [SEQ ID No: 11] encoding araCmev biosensor protein [SEQ ID No: 12] is located on the complementary strand to SEQ ID No: 7 at nucleotide positions: 96-974; **(1.sup.st) "FRT site" is located on complementary stand to SEQ ID No 13 at nucleotide positions: 247-281; (2.sup.nd) "FRT site" is located on complementary stand to SEQ ID No 13 at nucleotide positions: 1469-1503. The p.sub.BAD promoter is located a position 1241-1318 in SEQ ID No: 7.

[0106] In order to enhance to the stability of this plasmid based TA correction system, the native genomic copy of the yefM-yoeB gene pair was knocked-out. This was achieved using a DNA fragment comprising a FRT-kana-FRT resistance cassette fused to flanking sequences homologous to circa 200 bp flanking sequences homologous to each side of the junction between the chromosomal yefM-yoeB gene pair. Site specific integration of the FRT-kana-FRT resistance cassette into the native yefM-yoeB gene pair and functional knock-out was achieved by means of lambda-red-mediated recombineering.

1.2 Mevalonate Addiction Coupled to a Type II TA System is Shown to Control Bacterial Cell Growth

[0107] Mevalonate is one of the early precursors in the biosynthesis of a diverse group of compounds termed isoprenoids with a range of applications. The production of mevalonate in micro-organisms is seen to place a metabolic strain on the producing cells, meaning there is a fitness cost, and where the appearance of non-producing cells compromises the economics of mevalonate production. A Type II TA system coupled to mevalonate addiction according to the invention provides a method for slowing the growth or eliminating cells that are non-producers.

[0108] In order to control bacterial cell growth by mevalonate addiction, a mevalonate biosensor was introduced into the yefM-yoeB TA pair plasmid, pBAD-TA5 (Table 2), in place of the L-arabinose biosensor. The mevalonate-responsive biosensor was derived from the AraC sensor by introducing 4 point mutations (FIG. 4). In the absence of expression of the yefM antitoxin, cell growth was inhibited by the yoeB gene expressed under the control of the p.sub.LAC promoter. Addition of the inducer, IPTG, was not required, since the baseline activity of the Lac promoter was sufficient to drive expression of toxin at cell growth inhibitory levels; likely due to the leakiness of the Lac promoter.

[0109] Addition of mevalonate (37 mM) was sufficient to bind to the mevalonate biosensor and induce the cognate p.sub.BAD promoter to express the YefM antitoxin at levels sufficient to allow an increase in cell growth when compared to cell cultures devoid of mevalonate.

1.3 Engineering a Bacterial Strain Addicted to Internal Small Molecule Production

[0110] Mevalonate-addicted E. coli strains (as described in 1.2) were additionally engineered to produce mevalonate. Strains were transformed with a plasmid comprising genes encoding one of three alternative biosynthetic pathways for the conversion of endogenous acetyl CoA to mevalonate (FIG. 5). The plasmid, pMevTC, comprises an operon with the E. coli gene AtoB, encoding an acetyl-CoA acetyltransferase; an S. cerevisiae ERG13 gene encoding an HMG-CoA synthase, and tHMGR, a truncated version of S. cerevisiae HMGR, encoding an HMG-CoA reductase. This plasmid directs intermediate levels of mevalonate production in a host strain (1-2 g/L).

[0111] The plasmid, pMEV7C, comprises the E. coli gene AtoB; a Lactococcus lactis gene mvaS encoding an HMG-CoA synthase, and a Lactococcus lactis mvaE gene encoding a HMG-CoA reductase. This plasmid directs high level mevalonate production in a host strain (.ltoreq.14.6 g/L in batch fermentation).

[0112] A negative control pathway pMevT5c features a point mutation in the ERG13 gene, rendering the strain incapable of producing mevalonate, but otherwise it maintains the same promoter, enzymatic pathway reactions, antibiotic selection gene and plasmid origin of replication. The pathway operon promoter in both plasmids pMEV7C and pMevT5C was the synthetic promoter J23100 [SEQ ID No: 48].

[0113] Table 3 listing the genetic features of the mevalonate TA addicted strain m29 with internal mevalonate biosynthesis, based on the E. coli XL1 parent strain.

TABLE-US-00004 TABLE 3 Plasmid/ chromosome Gene SEQ ID No: Protein SEQ ID No pBAM-TA5 araCmev* SEQ ID No: p.sub.BAD-yefM 7 YefM SEQ ID No: 8 p.sub.Lac-yoeB YoeB SEQ ID No: 9 ampR Beta- SEQ ID No: 10 lactamase araCmev SEQ ID No: araCmev SEQ ID No: 12 11 chromosome yefM-FRT- SEQ ID No: Neomycin SEQ ID No: 14 kanR-FRT- 13 phospho- yoeB** transferase II pMEV7C atoB SEQ ID No: acetyl-CoA SEQ ID No: 16 15 acetyl- transferase mvaS HMG-CoA SEQ ID No: 17 synthase mvaE HMG-CoA SEQ ID No: 18 reductase camR*** camR SEQ ID No: Chloram- SEQ ID No: 20 19 phenicol acetyl- transferase *araCmev gene [SEQ ID No: 11] encoding araCmev biosensor protein [SEQ ID No: 12] is located on the complementary strand to SEQ ID No: 7 at nucleotide positions: 96-974; **(1.sup.st) "FRT site" is located on complementary stand to SEQ ID No 13 at nucleotide positions: 247-281; (2.sup.nd) "FRT site" is located on complementary stand to SEQ ID No 13 at nucleotide positions: 1469-1503. ***camR gene [SEQ ID No: 19] encoding chloramphenicol acetyltransferase [SEQ ID No: 20] is located on the complementary strand to SEQ ID No: 15 at nucleotide positions 4745-5404. The p.sub.BAD promoter is located a position 1241-1318 in SEQ ID No: 7.

[0114] Table 4 listing the genetic features of the mevalonate TA addicted strain m16 without internal mevalonate biosynthesis, based on the E. coli XL1 parent strain.

TABLE-US-00005 TABLE 4 Plasmid/chromosome Gene SEQ ID No: Protein SEQ ID No pBAM-TA5 araCmev* SEQ ID No: 7 p.sub.BAD-yefM YefM SEQ ID No: 8 p.sub.Lac-yoeB YoeB SEQ ID No: 9 ampR Beta-lactamase SEQ ID No: 10 araCmev SEQ ID No: 11 araCmev SEQ ID No: 12 chromosome yefM-FRT- SEQ ID No: 13 Neomycin SEQ ID No: 14 kanR-FRT- phosphotransferase yoeB** II pMevT5C atoB SEQ ID No: 21 acetyl-CoA SEQ ID No: 22 acetyltransferase Mutant Non-functional SEQ ID No: 23 ERG13 HMG-CoA synthase tHMGR Truncated HMG- SEQ ID No: 24 CoA reductase camR*** camR SEQ ID No: 19 Chloramphenicol SEQ ID No: 20 acetyltransferase *araCmev gene [SEQ ID No: 11] encoding araCmev biosensor protein [SEQ ID No: 12] is located on the complementary strand to SEQ ID No: 7 at nucleotide positions: 96-974; **(1.sup.st) "FRT site" located on complementary stand to SEQ ID No 13 at nucleotide positions: 247-281; (2.sup.nd) "FRT site" located on complementary stand to SEQ ID No 13 at nucleotide positions: 1469-1503. ***camR gene [SEQ ID No: 19] encoding chloramphenicol acetyltransferase [SEQ ID No: 20] is located on the complementary strand to SEQ ID No: 21 at nucleotide positions 5293-5952.

1.4 Mevalonate-Addiction Coupled to a TA System Cancels the Fitness Advantage of Non-Mevalonate Producing Cells.

[0115] Mevalonate production has a fitness cost for a bacterial cell. This is shown by the retarded growth of an E. coli strain, comprising the plasmid (pMEV7C) and producing `high` level mevalonate, as compared to an E. coli strain, comprising the control plasmid (pMevT5c) and producing no mevalonate, but still having the metabolic burden of expressing the enzymes of the inactivated mevalonate biosynthesis pathway (FIG. 6A). The product of HGMS activity is known to be toxic to the cell, and it is the accumulation of HMG-CoA which promotes growth inhibition.

[0116] Co-expression of the mevalonate-addiction TA system (pBAM-TA5), despite a longer lag phase in these cells, was effective in countering the fitness advantage of non-producing cells (FIG. 6B). Survival and growth of cells expressing the TA system (pBAM-TA5), depends on a sufficient level of mevalonate to induce expression of antitoxin. The rate of accumulation of internally synthesized mevalonate to levels sufficient to induce antitoxin production may be a factor contributing to the observed lag phase in producing cells that express mevalonate-addiction TA system.

1.5 Use of the TA System to Increase Small-Molecule Production by Bacterial Cultures Comprising Non-Producing Cells

[0117] The ability of the TA system to favor survival and growth of productive cells (i.e. cells producing the addiction molecule, mevalonate), was demonstrated by co-culturing mevalonate-producing cells, comprising the pMEV7C plasmid, and non-producing strains, comprising the pMevT5C plasmid. The ratios of non-producing:producing cells tested were: 0:100, 75:25 and 90:10. The total mevalonate production of the 3 co-cultured strains was tested with and without co-expression of the mevalonate-addiction TA system, encoded on the plasmid pBAM-TA5.

[0118] Previous experiments with strains possessing the mevalonate pathway had established a fitness cost of harboring a functional production plasmid (pMEV7c) versus a non-functional production plasmid (pMevT5c) to be a reduction in growth rate of approximately 20% (FIG. 6A). Accordingly, the growth of producing cells was by default expected to be disfavored compared to the non-producing cells (i.e. without any synthetic correction system). The same cell inoculum mixes were cultured with the producing and non-producing strains, but both also harbored the correction plasmid (pBAM-TA5). This addition of the correction system was shown to significantly enhance mevalonate production in the co-cultures comprising non-producing cells (FIG. 7).

Example 2 Essential Genes

2.1 L-Arabinose Addiction by Regulated Expression of an Essential Gene is Shown to Control Bacterial Cell Growth

[0119] The essential genes, used to control bacterial growth, are the E. coli genes folP and glmM, which are comprised together within a two-gene operon. The gene glmM encodes a phosphoglucosamine mutase; and the gene folP encodes a dihydropteroate synthase, which is part of the enzymatic pathway leading tetrahydrofolate (vitamin B9) synthesis, which is essential for normal cell growth.

[0120] The native single genomic copy of the essential gene operon folP, g/mM in E. coli host strain XL1, was modified to allow its transcriptional control by the L-arabinose/mevalonate-responsive promoter p.sub.BAD. Replacement of the native promoter by the p.sub.BAD promoter was engineered by introducing a knockout fragment containing a kanamycin resistance cassette and the p.sub.BAD promoter, as illustrated in the cartoon in FIG. 8. An L-arabinose biosensor (AraC) (Table 1) was introduced into the modified host strain on the plasmid pBAD18-cam. The modified and transformed E. coli strains were grown in 2.times.YT medium (10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl). Antibiotics were added to select for maintenance of plasmids in transformed strains, according to their respective antibiotic resistance gene. Antibiotic concentrations when added were as follows: ampicillin 100 .mu.g/mL, chloramphenicol 30 .mu.g/mL, spectinomycin 50 .mu.g/mL, kanamycin 50 .mu.g/mL. Table 5 lists the genetic features of the essential gene-based mevalonate-addicted strain e3.9, based on an E. coli XL1 parent strain. The chromosomally located sequence is an excerpt of the genome sequence that fully comprises the engineered features.

TABLE-US-00006 TABLE 5 Plasmid/ SEQ ID chromosome Gene No: Protein SEQ ID No chromosome (FRT-kanR- SEQ ID Dihydropteroate SEQ ID No: 26 FRT)-p.sub.BAD- No: 25 synthase folP* kanR** SEQ ID Neomycin SEQ ID No: 28 No: 27 phospho- transferase II pBAMspec araCmev*** SEQ ID specR No: 29 spectinomycin SEQ ID No: 30 resistance protein pMEV7C atoB SEQ ID acetyl-CoA SEQ ID No: 16 No: 15 acetyltransferase mvaS HMG-CoA SEQ ID No: 17 synthase mvaE HMG-CoA SEQ ID No: 18 reductase camR**** *The native promoter of the chromosomal folP-glmM operon is replaced by the p.sub.BAD promoter via the (FRT-KanR-FRT)-p.sub.BAD-folP cassette [SEQ ID No. 25]; which comprises the pBAD promoter at position 1907-1979 and a portion of the folP gene. The nucleotide sequence of the folP gene in the e3.9 strain is SEQ ID No: 87. **kanR gene [SEQ ID No: 27] encoding Neomycin phosphotransferase II [SEQ ID No: 28] is located on the complementary strand to SEQ ID No: 25 at nucleotide positions 389-1183. ***araCmev gene [SEQ ID No: 11] encoding araCmev biosensor protein [SEQ ID No: 12] is located on the complementary strand to SEQ ID No: 29 at nucleotide positions: 96-974. ****camR gene [SEQ ID No: 19] encoding chloramphenicol acetyltransferase [SEQ ID No: 20] is located on the complementary strand to SEQ ID No: 21 at nucleotide positions 4745-5404.

[0121] Table 6 listing the genetic features of the essential gene-based L-arabinose-addicted strain e3.5, based on an E. coli XL1 parent strain. The chromosomally located sequence is an excerpt of the genome sequence that fully comprises the engineered features.

TABLE-US-00007 TABLE 6 Plasmid/ SEQ ID chromosome Gene No: Protein SEQ ID No chromosome (FRT-kanR**- SEQ ID Dihydropteroate SEQ ID No: FRT)-p.sub.BAD- No: 25 synthase 26 folP* pBAD18- araC AraC cam*** cam Chloramphenicol acetyltransferase *The native promoter of the chromosomal folp-glmM operon is replaced by the p.sub.BAD promoter via the (FRT-KanR-FRT)-p.sub.BAD-folP cassette [SEQ ID No. 25]; which comprises the pBAD promoter at position 1907-1979 and a portion of the folP gene. The nucleotide sequence of the folp gene in the e3.5 strain is SEQ ID No: 87. **kanR gene [SEQ ID No: 27] encoding Neomycin phosphotransferase II [SEQ ID No: 28] is located on the complementary strand to SEQ ID No: 25 at nucleotide positions 389-1183. ***Guzman et al, 1995

[0122] Table 7 listing the genetic features of the pe1 control strain producing mevalonate without the addiction system, based on an E. coli XL1 parent strain.

TABLE-US-00008 TABLE 7 Plasmid/ SEQ ID chromosome Gene No: Protein SEQ ID No pBAM18-spec araCmev* SEQ ID AraCmev SEQ ID No: 12 specR No: 29 aminoglycoside SEQ ID No: 30 nucleotidyl- transferase pMEV7C atoB SEQ ID acetyl-CoA SEQ ID No: 16 No: 15 acetyltransferase mvaS HMG-CoA SEQ ID No: 17 synthase mvaE HMG-CoA SEQ ID No: 18 reductase camR** chloramphenicol SEQ ID No: 20 acetyltransferase *araCmev gene [SEQ ID No: 11] encoding araCmev biosensor protein [SEQ ID No: 12] is located on the complementary strand to SEQ ID No: 29 at nucleotide positions: 96-974; **camR gene [SEQ ID No: 19] encoding chloramphenicol acetyltransferase [SEQ ID No: 20] is located on the complementary strand to SEQ ID No: 21 at nucleotide positions 4745-5404; specR = spectomycin resistance protein (e.g. aminoglycoside nucleotidyltransferase).

[0123] Since the expression levels of essential genes is important for optimizing their use for growth control, a micro-library of four different ribosome binding sites (created by a single random base pair in one of the primers covering the RBS in front of the p.sub.BAD promoter), having different (RBS) strengths, were tested and one RBS was found to give a particularly good conditional growth, strictly in response to providing L-arabinose in the growth medium (FIG. 9). When cells, comprising the essential gene operon (folP/glmM) driven by the L-arabinose responsive p.sub.BAD promoter, were cultured in 2.times.YT growth medium supplemented with 0.25% L-arabinose at 37.degree. C., their growth was exponential, whereas cells with supplemented with only 0.0025% and 0% L-arabinose completely failed to grow over a 12-hour time course.

2.2 Use of Essential Genes to Enhance Small Molecule Production in Bacteria.

[0124] An E. coli strain comprising the essential gene operon folP-g/mM, whose expression was regulated by a mevalonate-addiction, was engineered in order to demonstrate its use for the regulation of mevalonate-dependent cell growth.

[0125] The native single genomic copy of the essential gene operon folP-g/mM in E. coli host strain XL1, was modified to allow its transcriptional control by the L-arabinose/mevalonate-responsive promoter p.sub.BAD (FIG. 8). A mevalonate biosensor (AraCmev) (Table 3) was introduced into the modified host strain on the plasmid pBAM 18-spec.

[0126] In order to demonstrate that growth and mevalonate production by the "essential gene" regulated E. coli strain was dependent on internal mevalonate production the cells were also transformed with a plasmid carrying the genes encoding the mevalonate biosynthetic pathway (pMEV7C (Table 2) or genes encoding the inactivated the mevalonate biosynthetic pathway (pMevT5C). Growth of these E. coli strains of producing strains (having a functional mevalonate pathway) was exponential, while growth of non-producing strains (having an inactivated mevalonate pathway) was strongly inhibited (FIG. 10).

2.3 Triacetic Acid Lactone Addiction by Regulated Expression of an Essential Gene is Shown to Control Bacterial Cell Growth

[0127] The native single genomic copy of the essential gene operon folP-g/mM in the E. coli XL1 host strain was modified to allow its transcriptional control by the triacetic acid lactone-responsive promoter p.sub.BAD. First, a gene encoding a triacetic acid lactone biosensor (AraCtal (Tang et al., 2013)) was introduced into the XL1 host strain on the plasmid pBALspec (Table 8). Then, replacement of the native folP-g/mM promoter in this host strain by the p.sub.BAD promoter was engineered by means of lambda red-mediated recombineering. Recombineering was performed using well-described methods utilizing the pKD46 plasmid (Datsenko and Wanner, 2000), but instead of gene disruption, a specific set of knock-out DNA fragments (produced by PCR) was used to only replace the native essential folP-g/mM operon promoter and folP ribosomal binding site (RBS). This set of knockout DNA fragments each contained a kanamycin resistance cassette and the p.sub.BAD promoter, as illustrated in the cartoon in FIG. 8. The nucleotide sequence of the set of gene knockout DNA fragments differed only with respect to the redundant nucleotide ("N", wherein "N" is any of A, T, G or C) in the RBS sequence (ACTTGC) for folP-g/mM, which generated a four-membered library of folP translational strengths. This library allowed selection of a translational strength for folP that resulted in control of normal cell growth in response to transcriptional regulation by the AraCtal biosensor.

TABLE-US-00009 TABLE 8 TAL responsive e3.16#5 clone Plasmid/ SEQ ID chromosome Gene No: Protein SEQ ID No chromosome (FRT-kanR- SEQ ID Dihydropteroate SEQ ID No: 26 FRT)-p.sub.BAD- No: 91* synthase folP* kanR** SEQ ID Neomycin SEQ ID No: 28 No: 27 phospho- transferase II pBALspec AraCtaI*** SEQ ID Triacetic acid SEQ ID No: 96 No: 93 lactone sensor protein specR spectinomycin SEQ ID No: 94 resistance protein *The native promoter of the chromosomal folP-glmM operon is replaced by the p.sub.BAD promoter via the (FRT-KanR-FRT)-p.sub.BAD-folp cassette [SEQ ID No. 91]; which comprises a p.sub.BAD promoter at position 1665-1990, a RBS at position 1980-1985, having the nucleotide sequence ACTTGC; and a portion of the folP gene. The nucleotide sequence of the complete folP gene in the e3.16#5 strain is SEQ ID No: 87. **kanR gene [SEQ ID No: 27] encoding Neomycin phosphotransferase II [SEQ ID No: 28] is located on the complementary strand to SEQ ID No: 91 at nucleotide positions 389-1183. ***araCtaI gene [SEQ ID No: 95] encoding araCtaI biosensor protein [SEQ ID No: 96] is located on the complementary strand to SEQ ID No: 93 at nucleotide positions: 96-1025. ****specR gene encoding spectinomycin resistance protein [SEQ ID No: 94] is located at nucleotide positions 1945-2916 of SEQ ID No: 93.

[0128] For recombineering, as performed in standard pKD46 protocols, the target strain was first transformed with pKD46, and a single colony of this strain was then cultured in a 25 mL 2.times.YT culture at 30 degrees C. and 250 rpm horizontal shaking. When the culture reached OD.sub.600=0.1, the lambda Red system of pKD46 was induced by addition of 0.2 vol % L-arabinose, and then further cultured to grow to OD.sub.600=0.4. The cells from the culture were then transformed with 300-500 ng knock-out DNA fragments by standard high-efficiency electroporation; the electroporated cells were then cultured in SOC medium (20 g/L tryptone, 5 g/L yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) at 37 degrees for at least 1-2 hours to recover and simultaneously cure the cells of the pKD46 plasmid. Following recovery, candidate recombineered cells were selected for by plating on LB agar plates supplemented with 10 mM triacetic acid lactone and spectinomycin for maintenance of pBALspec and kanamycin for selection of inserted knockout fragment.

[0129] A non-addicted control E. coli strain (e3.16CON), also equipped with spectinomycin and kanamycin resistance genes, but with wildtype control of folP-glmM expression (Table 9), was constructed in order to test the toxicity of triacetic acid lactone.

TABLE-US-00010 TABLE 9 Non-addicted control strain e3.16CON Plasmid/ SEQ ID chromosome Gene No: Protein SEQ ID No chromosome kanR SEQ ID Neomycin SEQ ID No: 28 No: 27 phospho- transferase II pBALspec AraCtaI*** SEQ ID Triacetic acid SEQ ID No: 96 No: 93 lactone sensor protein specR Spectinomycin SEQ ID No: 98 resistance protein ***araCtaI gene [SEQ ID No: 95] encoding araCtaI biosensor protein [SEQ ID No: 96] is located on the complementary strand to SEQ ID No: 93 at nucleotide positions: 96-1025.

[0130] The non-addicted control E. coli strain (e3.16CON), and a selected addicted strain (e3.16#5) were then tested and compared for triacetic acid lactone-dependent growth by cultivation in a microtiter plate reader in liquid 2.times.YT medium supplemented with spectinomycin and kanamycin and a three-step gradient of triacetic acid lactone (0, 2 and 20 mM). As seen in FIG. 11, the control strain showed a relative reduction in growth rate as a result of supplementation with triacetic acid lactone, indicating that triacetic acid lactone is toxic for growth. The observed growth depression was greatest at a concentration of 20 mM triacetic acid lactone.

[0131] The addicted strain e3.16#5 was able to grow in the absence of triacetic acid lactone, indicating that under non-inducing conditions the basal expression of the essential folP-glmM operon in this strain was sufficient to support growth.

[0132] Cell growth was however significantly reduced to a level matching the growth of the control strain under toxic condition of 20 mM triacetic acid lactone (FIG. 11 and FIG. 12). In contrast to the control strain, the growth rate of the addicted strain e3.16#5 was enhanced by the presence of 2 mM triacetic acid lactone, and the final cell yield was increased by the presence of both 2 mM and 20 mM triacetic acid lactone (FIG. 12). The dependence of the addicted strain on a supply of triacetic acid lactone for maximal growth (FIG. 12), confirms that the triacetic acid lactone biosensor is able to induce the p.sub.BAD promoter and the expression of dihydropteroate synthase, in the pathway leading tetrahydrofolate (vitamin B9) synthesis, which is essential for normal cell growth.

2.4. Salicylic Acid Addiction by Regulated Expression of an Essential Gene is Shown to Control Bacterial Cell Growth

[0133] The native single genomic copy of the essential gene operon folP-glmM in the E. coli XL1 host strain was modified to allow its transcriptional control by the salicylic acid-responsive promoter p.sub.SAL [SEQ ID No: 118]. First, a gene encoding a salicylic acid biosensor (Pseudomonas putida nahR_Asn169 (Cebolla et al., 1997)) was introduced into the XL1 host strain on the plasmid pBANspec (Table 10). Then, replacement of the native folP-g/mM promoter by the p.sub.SAL promoter was engineered by means of lambda red-mediated recombineering.

TABLE-US-00011 TABLE 10 Salicylic acid responsive e3.18#1 clone Plasmid/chromosome Gene SEQ ID No: Protein SEQ ID No chromosome (FRT-kanR- SEQ ID No: Dihydropteroate SEQ ID No: 102 FRT)-p.sub.SAL- 101 synthase folP* kanR** SEQ ID No: Neomycin SEQ ID No: 28 27 phosphotransferase II pBANspec NahRAsn169*** SEQ ID No: Salicylic acid SEQ ID No: 100 97 biosensor protein specR Spectinomycin SEQ ID No: 98 resistance protein *The native promoter of the chromosomal folP-glmM operon is replaced by the p.sub.SAL promoter via the (FRT-KanR-FRT)-p.sub.BAD-folP cassette [SEQ ID No. 101]; which comprises p.sub.SAL promoter at position 1794-1929; a RBS at position 1930-1935, having the nucleotide sequence ACTTGT; and a portion of the folP gene (to direct homologous recombination). The nucleotide sequence of the complete folP gene in the e3.18#1 strain is SEQ ID No: 87. **kanR gene [SEQ ID No: 27] encoding Neomycin phosphotransferase II [SEQ ID No: 28] is located on the complementary strand to SEQ ID No: 91 at nucleotide positions 389-1183. ***nahRAsn169 gene [SEQ ID No: 99] encoding salicylic acid biosensor protein [SEQ ID No: 100] is located on the complementary strand to SEQ ID No: 97 at nucleotide positions: 102-1004.

[0134] Recombineering was performed as described in section 2.3 using well-described methods utilizing the pKD46 plasmid (Datsenko and Wanner, 2000). Instead of gene disruption, a specific set of knock-out DNA fragments was used to only replace the native essential folP-glmM operon promoter and folP RBS. This set of knockout DNA fragments contained a kanamycin resistance cassette and the p.sub.SAL promoter, as illustrated in the cartoon in FIG. 8, as well as a redundant nucleotide ("N", wherein "N" is any of A, T, G or C) in the RBS for folP-glmM to generate a four-membered library of folP translational strengths. This library allows selection of a translational strength for folP that suits the transcriptional regulation by the biosensor. Finally, candidate recombineered cells were selected for by plating on LB agar plates supplemented with 5 mM salicylic acid and spectinomycin for maintenance of pBANspec; and kanamycin for selection of inserted knockout fragment. A control host strain e3.18CON (Table 11) also equipped with spectinomycin and kanamycin resistance genes, but with wild-type control of folP-glmM expression) was used to in order to test the toxicity of Salicylic acid.

TABLE-US-00012 TABLE 11 Non-addicted control strain e3.18CON Plasmid/chromosome Gene SEQ ID No: Protein SEQ ID No chromosome kanR SEQ ID No: 27 Neomycin SEQ ID No: 28 phosphotransferase II pBANspec NahRAsn169* SEQ ID No: 97 Salicylic acid SEQ ID No: 100 biosensor protein specR Spectinomycin SEQ ID No: 98 resistance protein *nahRAsn169 gene [SEQ ID No: 99] encoding salicylic biosensor protein [SEQ ID No: 100] is located on the complementary strand to SEQ ID No: 97 at nucleotide positions: 102-1004.

[0135] A selected addicted strain (e3.18#1) and the non-addicted control E. coli strain (e3.18CON) were then tested for salicylic acid dependent growth by cultivation in a microtiter plate reader in liquid 2.times.YT medium supplemented with spectinomycin and kanamycin and presence/absence of salicylic acid (0 or 5 mM). As seen in FIG. 13, the control strain showed a relative reduction in growth rate and final biomass yield as a result of supplementation with salicylic acid, indicating that salicylic acid is toxic for growth. In contrast to the control strain, the growth rate of the addicted strain e3.18#1 was not reduced by the presence of salicylic acid, but rather the growth rate was higher in presence of 5 mM salicylic acid than the otherwise expected rate for non-addicted strains (FIG. 14). The dependence of the addicted strain on a supply of salicylic acid for maximal growth (FIG. 14), confirms that the salicylic acid biosensor is able to induce the p.sub.SAL promoter and the expression of dihydropteroate synthase, in the pathway leading tetrahydrofolate (vitamin B9) synthesis, which is essential for normal cell growth.

2.5 Benzoic Acid Addiction by Regulated Expression of an Essential Gene is Shown to Control Bacterial Cell Growth

[0136] The native single genomic copy of the essential gene operon folP-glmM in the E. coli XL1 host strain was modified to allow its transcriptional control by the benzoic acid-responsive promoter p.sub.SAL [SEQ ID No: 118]. First, a gene encoding a benzoic acid biosensor (Pseudomonas putida nahR) (Table 12) was introduced into the XL1 host strain on the plasmid pBABspec. Then, replacement of the native folP-g/mM promoter by the p.sub.SAL promoter was engineered by means of lambda red-mediated recombineering.

TABLE-US-00013 TABLE 12 Benzoic acid addicted e3.22#4 clone Plasmid/chromosome Gene SEQ ID No: Protein SEQ ID No chromosome (FRT-kanR- SEQ ID Dihydropteroate SEQ ID No: 102 FRT)-p.sub.SAL- No: 101 synthase folP* kanR** SEQ ID No: 27 Neomycin SEQ ID No: 28 phosphotransferase II pBABspec NahR*** SEQ ID No: Benzoic acid SEQ ID No: 103 sensor protein 106 specR Spectinomycin SEQ ID No: resistance protein 104 *The native promoter of the chromosomal folP-glmM operon is replaced by the p.sub.SAL promoter via the (FRT-KanR-FRT)-p.sub.SAL-folp cassette [SEQ ID No. 101]; which comprises a p.sub.SAL promoter at position 1794-1929; a RBS at position 1930-1935, having the nucleotide sequence ACTTGC; and a portion of the folp gene. The nucleotide sequence of the complete folp gene in the e3.22#4 strain is SEQ ID No: 87. **kanR gene [SEQ ID No: 27] encoding Neomycin phosphotransferase II [SEQ ID No: 28] is located on the complementary strand to SEQ ID No: 91 at nucleotide positions 389-1183. ***nahR gene [SEQ ID No: 105] encoding benzoic biosensor protein [SEQ ID No: 106] is located on the complementary strand to SEQ ID No: 103 at nucleotide positions: 102-1004.

[0137] Recombineering was performed as described in section 2.3 using well-described methods utilizing the pKD46 plasmid (Datsenko and Wanner, 2000)). Instead of gene disruption, a specific set of knock-out DNA fragments was used to only replace the native essential folP-glmM operon promoter and folP RBS. This set of knockout DNA fragments contained a kanamycin resistance cassette and the p.sub.SAL promoter, as illustrated in the cartoon in FIG. 8, as well as a redundant nucleotide ("N", wherein "N" is any of A, T, G or C) in the RBS for folP-g/mM to generate a four-membered library of folP translational strengths. This library allows selection of a translational strength for folP that suits the transcriptional regulation by the biosensor.

[0138] A non-addicted control E. coli strain (e3.22CON), also equipped with spectinomycin and kanamycin resistance genes, but wild-type control of folP-glmM expression (Table 13), was constructed in order to test the toxicity of benzoic acid.

TABLE-US-00014 TABLE 13 Benzoic acid non-addicted control e3.22CON Plasmid/ SEQ ID chromosome Gene No: Protein SEQ ID No Chromosome kanR SEQ ID Neomycin SEQ ID No: 28 No: 27 phosphotransferase II pBABspec NahR SEQ ID Benzoic acid SEQ ID No: No: 103 sensor protein 106 specR Spectinomycin SEQ ID No: 104 resistance protein *kanR gene [SEQ ID No: 27] encoding Neomycin phosphotransferase II [SEQ ID No: 28]. ***nahR gene [SEQ ID No: 105] encoding benzoic biosensor protein [SEQ ID No: 106] is located on the complementary strand to SEQ ID No: 103 at nucleotide positions: 102-1004.

[0139] Finally, candidate recombineered cells were selected for by plating on LB agar plates supplemented with 5 mM benzoic acid and spectinomycin for maintenance of pBABspec and kanamycin for selection of inserted knockout fragment.

[0140] A selected addicted strain (e3.22#4) and the non-addicted control E. coli strain (e3.18CON) were then tested for benzoic acid dependent growth by cultivation in a microtiter plate reader in liquid 2.times.YT medium supplemented with spectinomycin and kanamycin and w/wo salicylic acid (0 or 5 mM). As seen in FIG. 15, the control strain showed a relative reduction in growth rate and final biomass yield as a result of supplementation with benzoic acid, indicating that benzoic acid is toxic for growth. In contrast to the control strain, the growth rate and final biomass yield of the addicted strain e3.18#1 was enhanced by supplementation with benzoic acid with a growth rate higher than otherwise expected for non-addicted strains (FIG. 16).

[0141] The dependence of the addicted strain on a supply of benzoic acid for maximal growth (FIG. 16), confirms that the benzoic acid biosensor is able to induce the p.sub.SAL promoter and the expression of dihydropteroate synthase, in the pathway leading tetrahydrofolate (vitamin B9) synthesis, which is essential for normal cell growth.

2.6 General Scheme for Engineering and Optimization of Addiction to Effector Metabolite by Regulated Expression of an Essential Gene

[0142] As exemplified in the previous examples, biosensors can be engineered into cells to control growth by addiction to an effector metabolite produced by the cell. The degree of addiction, which results in a relative growth advantage for cells that produce and accumulate the effector metabolite, may be increased by reducing the growth of an addicted cell in which the cellular levels of effector metabolite are below a given threshold. The degree of addiction of the addicted cell can be controlled by regulating the basal expression of the biosensor and/or the regulated essential gene.

[0143] 2.6.1 The degree of growth reduction for a given host cell that fails to produce sufficient effector metabolite can be increased by providing the regulated essential gene with a weaker RBS sequence e.g. generated with the "RBS Calculator" (https://salislab.net/software), thereby reducing its rate of translation. Alternatively, the basal transcription strength of the regulated essential gene can be reduced by providing a biosensor-regulated, but weaker, promoter to drive its expression. Inversely, the degree of addiction can be reduced, to minimize the growth reduction for non-producing cells, by employing a stronger RBS sequence or stronger promoter to enhance basal expression of the regulated essential gene.

2.6.2 The biosensor gene, engineered into the cell, is expressed under the control of a constitutive promoter. A suitable promoter can be selected from the set of the exemplary constitutive promoters provided in SEQ ID: 47-65. A suitable promoter is one that drives expression of the biosensor in an amount that is responsive to the effector metabolite concentration, while avoiding constitutive saturation of the promoter of the essential gene. Preferably, the engineered addicted cell of the invention is one whose growth rate is not reduced (no fitness cost) so long as the cell produces the effector metabolite at or above a predetermined amount.

2.7 Method for Engineering Host Cell Addiction to its Metabolic Product Using Tailor-Made Biosensor:Promoter Pairs

[0144] A large number of biosensors are described in the literature, in particular in the RegPrecise and Genbank databases, each being characterized for its specific detection of one of a wide range of metabolic products of interest, and which are suitable for engineering host cell addiction to a particular effector metabolite of interest.

[0145] By way of example, a list of suitable biosensor and cognate promoter pairs is given in Table 13 for respective effector metabolites, that are suitable for engineering host cell addiction, and for regulating the expression of a gene in the host cell required for its growth and/or survival. A host cell addicted to one of the effector metabolites listed in Table 13 can be constructed using the corresponding biosensor:promoter pairs to regulate the expression of the essential gene folP-glmM, as follows. The native folP-glmM promoter in the selected host strain is replaced by the respective promoter listed in Table 13, by means of lambda red-mediated recombineering as described in section 2.3 for insertion of the p.sub.BAD promoter. Further, the gene encoding the respective biosensor listed in Table 13, is introduced into the selected host strain on a plasmid, as described for the introduction of the plasmid pBALspec comprising the triacetic acid lactone biosensor in Example 2.3. Standard molecular expression tuning may additionally be employed to optimize the degree of addiction as described above.

TABLE-US-00015 TABLE 13 Specific implementations of biosensors for diverse effectors Biosensor Biosensor Effector Effector Promoter Biosensor nucleotide protein metabolite metabolite Promoter name name sequence sequence conc. (.mu.M) Phenol SEQ ID No: 107 dmpRKLM DmpR SEQ No: 108 SEQ No: 109 1000 2-Amino- SEQ No: 110 hbpC HbpR SEQ No: 111 SEQ No: 112 100 biphenol Catechol SEQ No: 107 dmpRKLM CapR SEQ No: 113 SEQ No: 114 100 D-glycerate SEQ No: 115 gudP CdaR SEQ No: 116 SEQ No: 117 750 triacetic acid SEQ ID No: 66 p.sub.BAD AraCtal SEQ ID No: 95 SEQ ID No: 96 lactone salicylic acid SEQ ID No: 118 p.sub.SAL NahR_Asn169 SEQ ID No: 99 SEQ ID No: 100 benzoic acid SEQ ID No: 118 p.sub.SAL NahR SEQ ID No: 105 SEQ ID No: 106

Example 3. Riboswitch Biosensors Extend the Range of Addiction Molecules for Regulating Cell Growth

3.1 Use of Thiamine Pyrophosphate (TPP)-Sensitive Riboswitch-Controlled TA System to Regulate Bacterial Cell Growth

[0146] A TPP-responsive riboswitch (ThiMN15#19) was engineered into the previously constructed L-arabinose-responsive TA-based correction system (pBAD-TA5 to yield pBAT-TA5). This TPP riboswitch functions as an "ON-type" when in the presence of its ligand TPP, and it is functional at the level of translation. The riboswitch was inserted into the DNA region of the pBAD-TA5 gene construct that encodes the 5' untranslated region (UTR) of the antitoxin mRNA sequence (FIG. 17). For optimal functionality of the riboswitch, a protein-encoding sequence tet was also added as N-terminal fusion to YefM as it originated from the screen where ThiMN15#19 had been identified. An alternative TPP-responsive riboswitch was constructed, where an "OFF-type" riboswitch was inserted into the DNA region of the pBAD-TA5 gene construct that encodes the 5'-UTR of the toxin-encoding sequence to yield the same ligand-responsive growth.

[0147] Table 14 listing the genetic features of the TPP-addicted-TA-regulated E. coli strain based on the E. coli XL1 parent strain

TABLE-US-00016 TABLE 14 Plasmid/ chromosome Gene SEQ ID No: Protein SEQ ID No pBAT-TA5 araC* SEQ ID No: 31 AraC Thi-tet- Tet-YefM SEQ ID No: 32 yefM** yoeB YoeB SEQ ID No: 33 ampR Beta- SEQ ID No: 34 lactamase *araC gene [SEQ ID No: 5] encoding araC biosensor protein [SEQ ID No: 6] is located on the complementary strand to SEQ ID No: 31 at nucleotide positions: 96-974; **thi riboswitch is located at nucleotides 1291-1452 of SEQ ID No.: 31

[0148] A modified E. coli strain comprising the TPP responsive-riboswitch-regulated TA correction system (pBAT-TA5) showed enhanced growth in the presence of the inducer TPP, thereby demonstrating functionality of the TPP-addiction mediated via a riboswitch and TA system (FIG. 18). An alternative TPP responsive-riboswitch-regulated TA correction system was engineered with a constitutive promoter driving expression of the riboswitch-yefM mRNA. From a selection of constitutive promoters (SEQ ID Nos.: 47-65, 89) of different strengths, the p.sub.BAD promoter of pBAT-TA5 was replaced and the resulting plasmids transformed into E. coli XL1. These were tested to identify plasmids where the strength of the constitutive promoter allowed the riboswitch-yefM transcript to cause good TPP-conditional growth of the transformed E. coli cell, i.e. when grown in liquid medium with and without addition of 500 .mu.M TPP.

3.1 Use of a TPP-Sensitive Riboswitch-Regulated TA System to Enhance Small Molecule Production in Bacteria.

[0149] In order to demonstrate that modified E. coli strain comprising the TPP riboswitch (ThiMN15#19) TA system, was dependent on internal TPP overproduction, the cells were further genetically modified to biosynthesize higher concentrations of TPP, which were compared with a reference strain cells retaining the wild-type intracellular TPP accumulation level. Biosynthesis of elevated TPP concentration was engineered by translational deregulation of the chromosomal native E. coli thiC gene. This was achieved by introducing a point mutation (by substituting wild-type nucleotide residue A at position (-135), with nucleotide B (=any one of C, G and T) in the region of the native E. coli thiC gene [GeneID: 948492], to give the following 142nt sequence located directly upstream of the thiC open reading frame, corresponding to the transcribed 5'-UTR: ATTCGGGBTCCGCGGAACCTGATCAGGCTAATACCTGCGAAGGGAACAAGAGTTAATCTGCTA TCGCATCGCCCCTGCGGCGATCGTCTCTTGCTTCATCCGTCGTCTGACAAGCCACGTCCTTAA CTTTTTGGAATGAGCT [SEQ ID No: 90] which when transcribed yields a mutant E. coli riboswitch transcript, that is insensitive to TPP feedback regulation. Growth and TPP production by these E. coli strains comprising, or lacking, the TPP-sensitive riboswitch-regulated TA gene correction system in the modified and reference stains, are then compared.

Example 4 Improved Fermentation Productivity Through Use of Product-Addicted Strain Based on Control of Essential Genes

[0150] A plasmid (pMEV7C), encoding the genes for the metabolic pathway to mevalonate, was inserted in E. coli XL1. The strain was further engineered to encompass a biosensor-based addiction module according to the invention e.g. by following the methods for construction of strain e3.9 comprising the plasmid (FRT-kana-FRT-p.sub.BAD-RBS1)-folP-glmM), where growth requires expression of an essential gene (see Example 2.2). As a control, the same pathway plasmid (pMEV7C) was inserted in an E. coli XL1 strain to generate pe1 only differing in way of genetic engineering from e3.9 (Table 3) by the fact that no genetic changes had been introduced on the chromosome (i.e. the expression of essential genes were not linked to presence of the metabolic pathway product).

[0151] The two strains e3.9 and pe1 were grown at 37 deg. C. with 250-300 rpm horizontal shaking in 2.times.YT medium (with 30 .mu.g/mL chloramphenicol and 50 .mu.g/mL spectinomycin) for 55 cell generations to simulate a fermentation of large industrial size. This generation number was obtained in 25 mL shake flask cultures by transferring .gtoreq.0.5 vol % culture to fresh 2.times.YT medium an appropriate number of times according to the cell densities measured when transferring the culture. Final productivity of the cell population was evaluated by taking a sample for HPLC analysis following culturing at 37 deg. C. for 72 hours after the final transferring. As seen in FIG. 19, the concentration of mevalonate (MVA) in the control strain pe1 was only 35% of the concentration in the e3.9 strain, demonstrating the advantage of linking product-presence to growth of the strain during cultivations with many cell divisions.

[0152] A high generation number could be reached using a continuous chemostat fermentor, in which the cells are constantly dividing through inlet of new medium and outlet of culture. Optionally, the strains could also be cultured at another temperature such as 30 deg. C. The productivity of the cells could further be measured at various numbers of generations such as (but not limited to) 20, 30, 40, 50, 60, 70 and 150 generations. To evaluate the stability of the pathway over time (cell generations), the cells can be re-cultured from a stored sample of the generation of cells in a 25% glycerol stock at -80 degrees Celsius. This sample could be re-cultured in 15 mL 2.times.YT (with 30 .mu.g/mL chloramphenicol and 50 .mu.g/mL spectinomycin) under otherwise same culture conditions and measuring the productivity (according to method described in previous section). To evaluate whether genetic mutations were less predominant in the product pathway of the product-addicted strain, DNA sequencing of the strains could be performed.

[0153] The cultivations can be carried out in growth medium, which can direct higher mevalonate productivity, such as M9 minimal medium supplemented with 0.4-4 (w/v) % glucose.

[0154] Mevalonate production (in the culture medium) was detected by HPLC, following treatment of 300 .mu.L sampled culture broth with 23 .mu.L 20% sulfuric acid for conversion to the mevalonolactone form. The mixture was vortexed and cells separated from the medium by centrifugation at 17,000 g for 3 mins. 30 .mu.L supernatant (medium) was injected in an Ultimate 3000 HPLC running with a 5 mM sulfuric acid mobile phase (0.6 mL/min) on an Aminex HPX-87H ion exclusion column (300 mm.times.7.8 mm, Bio-Rad Laboratories, Hercules, Calif., USA) at 50.degree. C., with detection using refractive-index (RI) channel.

Example 5 Improved Fermentation Productivity Through Use of Product-Addicted Strain Based on Control of Toxin-Antitoxin Systems

[0155] A plasmid (pMEV7C) encoding the genes for the metabolic pathway to mevalonate was inserted in E. coli XL1. The strain was further engineered to encompass a biosensor-based addiction module according to the invention e.g. by following the methods for construction of strain m29, which comprises (pBAM-TA5), where growth is controlled by expression of the anti-toxin and toxin pair (see Example 1.4). As a control, the exact same pathway plasmid was inserted in an E. coli XL1 strain, to create a strain only differing from m29 by the fact that a plasmid featuring the invented system had not been introduced (i.e. the expression of antitoxin was not linked to presence of the metabolic pathway product).

[0156] Pre-cultures in 2.times.YT were inoculated from single colonies of the two strains, and main cultures were inoculated from these when OD600 (measured on 200 .mu.L sample in a BioTek SynergyH1 plate reader) was 0.1-0.5. From there, the two strains were grown at 30 deg. C. with 250-300 rpm horizontal shaking in 2.times.YT medium for 70 cell generations to simulate a fermentation of large industrial size. This generation number was obtained in 50 mL shake flask cultures by transferring .gtoreq.0.5 vol % culture to fresh medium an appropriate number of times according to the cell densities measured before transferring the culture. Optionally, the generation number could be reached using a continuous chemostat fermentor, in which the cells are constantly dividing through inlet of new medium and outlet of culture. The productivity of the cells was measured at various numbers of generations such as (but not limited to) 20, 30, 40, 50, 60 and 70 generations. This was done by recording a sample of the generation of cells in a 25% glycerol stock stored at -80 deg. C. and re-culturing them in tubes with 15 mL 2.times.YT under otherwise same culture conditions and measuring the productivity (according to method described in Example 4).

REFERENCES

[0157] Cebolla, A., Sousa, C., De Lorenzo, V., 1997. Effector Specificity Mutants of the Transcriptional Activator NahR of Naphthalene Degrading Pseudomonas Define Protein Sites Involved in Binding of Aromatic Inducers. J. Biol. Chem. 272, 3986-3992. doi:10.1074/jbc.272.7.3986

[0158] Datsenko, K. a, Wanner, B. L., 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A 97, 6640-6645. doi:10.1073/pnas.120163297

[0159] Guzman, L. M., Belin, D., Carson, M. J., & Beckwith, J. (1995): Tight regulation, modulation, and high-level expression by vectors containing the arabinose P BAD Promoter. Journal of Bacteriology, 177(14), 4121-4130.

[0160] Tang, S. Y., Qian, S., Akinterinwa, O., Frei, C. S., Gredell, J. A., Cirino, P. C., 2013. Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter. J. Am. Chem. Soc. 135, 10099-10103. doi:10.1021/ja402654z

Sequence CWU 1

1

11815384DNAEscherichia colipromoter(1246)..(1318)pBAD promoterCDS(1330)..(1581)yefM antitoxin proteinCDS(1845)..(2099)yeoB toxin proteinCDS(2652)..(3512)AmpR encoded beta-lactamase 1atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga 720tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcgaatt cgagctcgag 1320gaggaaggt atg cgt aca att agc tac agc gaa gcg cgt cag aat ttg tcg 1371 Met Arg Thr Ile Ser Tyr Ser Glu Ala Arg Gln Asn Leu Ser 1 5 10 gca aca atg atg aaa gcc gtt gaa gat cat gcc ccg atc ctt att act 1419Ala Thr Met Met Lys Ala Val Glu Asp His Ala Pro Ile Leu Ile Thr 15 20 25 30 cgt cag aat gga gag gct tgt gtt ctg atg tca ctc gaa gaa tac aac 1467Arg Gln Asn Gly Glu Ala Cys Val Leu Met Ser Leu Glu Glu Tyr Asn 35 40 45 tcg ctg gaa gag acg gct tat cta ctg cgc tcc ccc gct aac gcc cgg 1515Ser Leu Glu Glu Thr Ala Tyr Leu Leu Arg Ser Pro Ala Asn Ala Arg 50 55 60 aga ttg atg gac tca atc gat agc ctg aaa tca ggc aaa gga acg gaa 1563Arg Leu Met Asp Ser Ile Asp Ser Leu Lys Ser Gly Lys Gly Thr Glu 65 70 75 aag gac atc att gag tga ggcaatcagc tgttgcccgt ctcactggtg 1611Lys Asp Ile Ile Glu 80 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 1671tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 1731aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 1791tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaagaa acc atg 1847 Met aaa cta atc tgg tct gag gaa tca tgg gac gat tat ctg tac tgg cag 1895Lys Leu Ile Trp Ser Glu Glu Ser Trp Asp Asp Tyr Leu Tyr Trp Gln 85 90 95 100 gaa aca gat aag cga att gtt aaa aag atc aat gaa ctt atc aaa gat 1943Glu Thr Asp Lys Arg Ile Val Lys Lys Ile Asn Glu Leu Ile Lys Asp 105 110 115 acc cgc aga acg cca ttt gaa ggt aag ggg aag cca gaa ccc ctg aaa 1991Thr Arg Arg Thr Pro Phe Glu Gly Lys Gly Lys Pro Glu Pro Leu Lys 120 125 130 cat aat ttg tca ggt ttc tgg tcc cga cgc att aca gag gag cac cgt 2039His Asn Leu Ser Gly Phe Trp Ser Arg Arg Ile Thr Glu Glu His Arg 135 140 145 ctg gta tac gcg gtt acc gac gat tca ctg ctc att gca gca tgt cgt 2087Leu Val Tyr Ala Val Thr Asp Asp Ser Leu Leu Ile Ala Ala Cys Arg 150 155 160 tat cat tat tga atcctctaga gtcgacctgc aggcatgcaa gcttggctgt 2139Tyr His Tyr 165 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt 2199ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg 2259aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta 2319gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 2379tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt 2439gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag 2499gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct 2559tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 2619aaatgcttca ataatattga aaaaggaaga gt atg agt att caa cat ttc cgt 2672 Met Ser Ile Gln His Phe Arg 170 gtc gcc ctt att ccc ttt ttt gcg gca ttt tgc ctt cct gtt ttt gct 2720Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala 175 180 185 190 cac cca gaa acg ctg gtg aaa gta aaa gat gct gaa gat cag ttg ggt 2768His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly 195 200 205 gca cga gtg ggt tac atc gaa ctg gat ctc aac agc ggt aag atc ctt 2816Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu 210 215 220 gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc act ttt aaa 2864Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys 225 230 235 gtt ctg cta tgt ggc gcg gta tta tcc cgt gtt gac gcc ggg caa gag 2912Val Leu Leu Cys Gly Ala Val Leu Ser Arg Val Asp Ala Gly Gln Glu 240 245 250 caa ctc ggt cgc cgc ata cac tat tct cag aat gac ttg gtt gag tac 2960Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr 255 260 265 270 tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa 3008Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu 275 280 285 tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta 3056Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu 290 295 300 ctt ctg aca acg atc gga gga ccg aag gag cta acc gct ttt ttg cac 3104Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His 305 310 315 aac atg ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg 3152Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu 320 325 330 aat gaa gcc ata cca aac gac gag cgt gac acc acg atg cct gca gca 3200Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Ala Ala 335 340 345 350 atg gca aca acg ttg cgc aaa cta tta act ggc gaa cta ctt act cta 3248Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu 355 360 365 gct tcc cgg caa caa tta ata gac tgg atg gag gcg gat aaa gtt gca 3296Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala 370 375 380 gga cca ctt ctg cgc tcg gcc ctt ccg gct ggc tgg ttt att gct gat 3344Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp 385 390 395 aaa tct gga gcc ggt gag cgt ggg tct cgc ggt atc att gca gca ctg 3392Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu 400 405 410 ggg cca gat ggt aag ccc tcc cgt atc gta gtt atc tac acg acg ggg 3440Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly 415 420 425 430 agt cag gca act atg gat gaa cga aat aga cag atc gct gag ata ggt 3488Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly 435 440 445 gcc tca ctg att aag cat tgg taa ctgtcagacc aagtttactc atatatactt 3542Ala Ser Leu Ile Lys His Trp 450 tagattgatt tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 3602cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 3662ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 3722gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc 3782acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 3842ctttaatagt ggactcttgt tccaaacttg aacaacactc aaccctatct cgggctattc 3902ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 3962acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttaaa aggatctagg 4022tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 4082gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 4142taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 4202aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 4262ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 4322catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 4382ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 4442ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 4502agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 4562taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 4622atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 4682cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 4742ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 4802accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 4862gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 4922tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 4982agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca 5042cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 5102acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 5162acgcgcgagg cagcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca 5222tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg 5282tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga 5342tgcgtccggc gtagaggatc tgctcatgtt tgacagctta tc 5384283PRTEscherichia coli 2Met Arg Thr Ile Ser Tyr Ser Glu Ala Arg Gln Asn Leu Ser Ala Thr 1 5 10 15 Met Met Lys Ala Val Glu Asp His Ala Pro Ile Leu Ile Thr Arg Gln 20 25 30 Asn Gly Glu Ala Cys Val Leu Met Ser Leu Glu Glu Tyr Asn Ser Leu 35 40 45 Glu Glu Thr Ala Tyr Leu Leu Arg Ser Pro Ala Asn Ala Arg Arg Leu 50 55 60 Met Asp Ser Ile Asp Ser Leu Lys Ser Gly Lys Gly Thr Glu Lys Asp 65 70 75 80 Ile Ile Glu 384PRTEscherichia coli 3Met Lys Leu Ile Trp Ser Glu Glu Ser Trp Asp Asp Tyr Leu Tyr Trp 1 5 10 15 Gln Glu Thr Asp Lys Arg Ile Val Lys Lys Ile Asn Glu Leu Ile Lys 20 25 30 Asp Thr Arg Arg Thr Pro Phe Glu Gly Lys Gly Lys Pro Glu Pro Leu 35 40 45 Lys His Asn Leu Ser Gly Phe Trp Ser Arg Arg Ile Thr Glu Glu His 50 55 60 Arg Leu Val Tyr Ala Val Thr Asp Asp Ser Leu Leu Ile Ala Ala Cys 65 70 75 80 Arg Tyr His Tyr 4286PRTEscherichia coli 4Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30 Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp 35 40 45 Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 50 55 60 Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 65 70 75 80 Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 85 90 95 Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 100 105 110 Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser 115 120 125 Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys 130 135 140 Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu 145 150 155 160 Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg 165 170 175 Asp Thr Thr Met Pro Ala Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 180 185 190 Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp 195 200 205 Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 210 215 220 Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser 225 230 235 240 Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile 245 250 255 Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn 260 265 270 Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp 275 280 285 5879DNAEscherichia coliCDS(1)..(879)araC arabinose sensor protein 5atg gct gaa gcg caa aat gat ccc ctg ctg ccg gga tac tcg ttt aat 48Met Ala Glu Ala Gln Asn Asp Pro Leu Leu Pro Gly Tyr Ser Phe Asn 1 5 10 15 gcc cat ctg gtg gcg ggt tta acg ccg att gag gcc aac ggt tat ctc 96Ala His Leu Val Ala Gly Leu Thr Pro Ile Glu Ala Asn Gly Tyr Leu 20 25 30 gat ttt ttt atc gac cga ccg ctg gga atg aaa ggt tat att ctc aat 144Asp Phe Phe Ile Asp Arg Pro Leu Gly Met Lys Gly Tyr Ile Leu Asn 35 40 45 ctc acc att cgc ggt cag ggg gtg gtg aaa aat cag gga cga gaa ttt 192Leu Thr Ile Arg Gly Gln Gly Val Val Lys Asn Gln Gly Arg Glu Phe 50 55 60 gtt tgc cga ccg ggt gat att ttg ctg ttc ccg cca gga gag att cat 240Val Cys Arg Pro Gly Asp Ile Leu Leu Phe Pro Pro Gly Glu Ile His 65 70 75 80 cac tac ggt cgt cat ccg gag gct cgc gaa tgg tat cac cag tgg gtt 288His Tyr Gly Arg His Pro Glu Ala Arg Glu Trp Tyr His Gln Trp Val 85 90 95 tac ttt cgt ccg cgc gcc tac tgg cat gaa tgg ctt aac tgg ccg tca 336Tyr Phe Arg Pro Arg Ala Tyr Trp His Glu Trp Leu Asn Trp Pro Ser 100 105 110 ata ttt gcc aat acg ggg ttc ttt cgc ccg gat gaa gcg cac cag ccg 384Ile Phe Ala Asn Thr Gly Phe Phe Arg Pro Asp Glu Ala His Gln Pro 115 120 125 cat ttc agc gac ctg ttt ggg caa atc att aac gcc ggg caa ggg gaa 432His Phe Ser Asp Leu Phe Gly Gln Ile Ile Asn Ala Gly Gln Gly Glu 130 135 140 ggg cgc tat tcg gag ctg ctg gcg ata aat ctg ctt gag caa ttg tta 480Gly Arg Tyr Ser Glu Leu Leu Ala Ile Asn Leu Leu Glu Gln Leu Leu 145 150 155 160 ctg cgg cgc atg gaa gcg att aac gag tcg ctc cat cca ccg atg gat 528Leu Arg Arg Met Glu Ala Ile Asn Glu Ser Leu His Pro Pro Met Asp 165 170 175 aat cgg gta cgc gag gct tgt cag tac atc agc gat cac ctg gca gac 576Asn Arg Val Arg Glu Ala Cys Gln Tyr Ile Ser Asp His Leu Ala Asp 180 185 190 agc aat ttt gat atc gcc agc gtc gca cag cat gtt tgc ttg tcg ccg 624Ser Asn Phe Asp Ile Ala Ser Val Ala Gln His Val Cys Leu Ser Pro 195 200 205

tcg cgt ctg tca cat ctt ttc cgc cag cag tta ggg att agc gtc tta 672Ser Arg Leu Ser His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val Leu 210 215 220 agc tgg cgc gag gac caa cgt atc agc cag gcg aag ctg ctt ttg agc 720Ser Trp Arg Glu Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu Ser 225 230 235 240 acc acc cgg atg cct atc gcc acc gtc ggt cgc aat gtt ggt ttt gac 768Thr Thr Arg Met Pro Ile Ala Thr Val Gly Arg Asn Val Gly Phe Asp 245 250 255 gat caa ctc tat ttc tcg cgg gta ttt aaa aaa tgc acc ggg gcc agc 816Asp Gln Leu Tyr Phe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala Ser 260 265 270 ccg agc gag ttc cgt gcc ggt tgt gaa gaa aaa gtg aat gat gta gcc 864Pro Ser Glu Phe Arg Ala Gly Cys Glu Glu Lys Val Asn Asp Val Ala 275 280 285 gtc aag ttg tca taa 879Val Lys Leu Ser 290 6292PRTEscherichia coli 6Met Ala Glu Ala Gln Asn Asp Pro Leu Leu Pro Gly Tyr Ser Phe Asn 1 5 10 15 Ala His Leu Val Ala Gly Leu Thr Pro Ile Glu Ala Asn Gly Tyr Leu 20 25 30 Asp Phe Phe Ile Asp Arg Pro Leu Gly Met Lys Gly Tyr Ile Leu Asn 35 40 45 Leu Thr Ile Arg Gly Gln Gly Val Val Lys Asn Gln Gly Arg Glu Phe 50 55 60 Val Cys Arg Pro Gly Asp Ile Leu Leu Phe Pro Pro Gly Glu Ile His 65 70 75 80 His Tyr Gly Arg His Pro Glu Ala Arg Glu Trp Tyr His Gln Trp Val 85 90 95 Tyr Phe Arg Pro Arg Ala Tyr Trp His Glu Trp Leu Asn Trp Pro Ser 100 105 110 Ile Phe Ala Asn Thr Gly Phe Phe Arg Pro Asp Glu Ala His Gln Pro 115 120 125 His Phe Ser Asp Leu Phe Gly Gln Ile Ile Asn Ala Gly Gln Gly Glu 130 135 140 Gly Arg Tyr Ser Glu Leu Leu Ala Ile Asn Leu Leu Glu Gln Leu Leu 145 150 155 160 Leu Arg Arg Met Glu Ala Ile Asn Glu Ser Leu His Pro Pro Met Asp 165 170 175 Asn Arg Val Arg Glu Ala Cys Gln Tyr Ile Ser Asp His Leu Ala Asp 180 185 190 Ser Asn Phe Asp Ile Ala Ser Val Ala Gln His Val Cys Leu Ser Pro 195 200 205 Ser Arg Leu Ser His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val Leu 210 215 220 Ser Trp Arg Glu Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu Ser 225 230 235 240 Thr Thr Arg Met Pro Ile Ala Thr Val Gly Arg Asn Val Gly Phe Asp 245 250 255 Asp Gln Leu Tyr Phe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala Ser 260 265 270 Pro Ser Glu Phe Arg Ala Gly Cys Glu Glu Lys Val Asn Asp Val Ala 275 280 285 Val Lys Leu Ser 290 75384DNAEscherichia colipromoter(1241)..(1318)pBAD promoterCDS(1330)..(1581)yefM anti-toxin geneCDS(1845)..(2099)yoeB toxin proteinCDS(2652)..(3512)AmpR encoded beta-lactamase 7atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660tgccagtagg cgcgcggacg aaagtaaacc cactgtcgat accattcgcg agcctccgga 720tgacgaccca agtgcagaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900gggagtaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcgaatt cgagctcgag 1320gaggaaggt atg cgt aca att agc tac agc gaa gcg cgt cag aat ttg tcg 1371 Met Arg Thr Ile Ser Tyr Ser Glu Ala Arg Gln Asn Leu Ser 1 5 10 gca aca atg atg aaa gcc gtt gaa gat cat gcc ccg atc ctt att act 1419Ala Thr Met Met Lys Ala Val Glu Asp His Ala Pro Ile Leu Ile Thr 15 20 25 30 cgt cag aat gga gag gct tgt gtt ctg atg tca ctc gaa gaa tac aac 1467Arg Gln Asn Gly Glu Ala Cys Val Leu Met Ser Leu Glu Glu Tyr Asn 35 40 45 tcg ctg gaa gag acg gct tat cta ctg cgc tcc ccc gct aac gcc cgg 1515Ser Leu Glu Glu Thr Ala Tyr Leu Leu Arg Ser Pro Ala Asn Ala Arg 50 55 60 aga ttg atg gac tca atc gat agc ctg aaa tca ggc aaa gga acg gaa 1563Arg Leu Met Asp Ser Ile Asp Ser Leu Lys Ser Gly Lys Gly Thr Glu 65 70 75 aag gac atc att gag tga ggcaatcagc tgttgcccgt ctcactggtg 1611Lys Asp Ile Ile Glu 80 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 1671tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 1731aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 1791tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaagaa acc atg 1847 Met aaa cta atc tgg tct gag gaa tca tgg gac gat tat ctg tac tgg cag 1895Lys Leu Ile Trp Ser Glu Glu Ser Trp Asp Asp Tyr Leu Tyr Trp Gln 85 90 95 100 gaa aca gat aag cga att gtt aaa aag atc aat gaa ctt atc aaa gat 1943Glu Thr Asp Lys Arg Ile Val Lys Lys Ile Asn Glu Leu Ile Lys Asp 105 110 115 acc cgc aga acg cca ttt gaa ggt aag ggg aag cca gaa ccc ctg aaa 1991Thr Arg Arg Thr Pro Phe Glu Gly Lys Gly Lys Pro Glu Pro Leu Lys 120 125 130 cat aat ttg tca ggt ttc tgg tcc cga cgc att aca gag gag cac cgt 2039His Asn Leu Ser Gly Phe Trp Ser Arg Arg Ile Thr Glu Glu His Arg 135 140 145 ctg gta tac gcg gtt acc gac gat tca ctg ctc att gca gca tgt cgt 2087Leu Val Tyr Ala Val Thr Asp Asp Ser Leu Leu Ile Ala Ala Cys Arg 150 155 160 tat cat tat tga atcctctaga gtcgacctgc aggcatgcaa gcttggctgt 2139Tyr His Tyr 165 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt 2199ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg 2259aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta 2319gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 2379tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt 2439gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag 2499gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct 2559tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 2619aaatgcttca ataatattga aaaaggaaga gt atg agt att caa cat ttc cgt 2672 Met Ser Ile Gln His Phe Arg 170 gtc gcc ctt att ccc ttt ttt gcg gca ttt tgc ctt cct gtt ttt gct 2720Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala 175 180 185 190 cac cca gaa acg ctg gtg aaa gta aaa gat gct gaa gat cag ttg ggt 2768His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly 195 200 205 gca cga gtg ggt tac atc gaa ctg gat ctc aac agc ggt aag atc ctt 2816Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu 210 215 220 gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc act ttt aaa 2864Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys 225 230 235 gtt ctg cta tgt ggc gcg gta tta tcc cgt gtt gac gcc ggg caa gag 2912Val Leu Leu Cys Gly Ala Val Leu Ser Arg Val Asp Ala Gly Gln Glu 240 245 250 caa ctc ggt cgc cgc ata cac tat tct cag aat gac ttg gtt gag tac 2960Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr 255 260 265 270 tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa 3008Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu 275 280 285 tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta 3056Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu 290 295 300 ctt ctg aca acg atc gga gga ccg aag gag cta acc gct ttt ttg cac 3104Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His 305 310 315 aac atg ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg 3152Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu 320 325 330 aat gaa gcc ata cca aac gac gag cgt gac acc acg atg cct gca gca 3200Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Ala Ala 335 340 345 350 atg gca aca acg ttg cgc aaa cta tta act ggc gaa cta ctt act cta 3248Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu 355 360 365 gct tcc cgg caa caa tta ata gac tgg atg gag gcg gat aaa gtt gca 3296Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala 370 375 380 gga cca ctt ctg cgc tcg gcc ctt ccg gct ggc tgg ttt att gct gat 3344Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp 385 390 395 aaa tct gga gcc ggt gag cgt ggg tct cgc ggt atc att gca gca ctg 3392Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu 400 405 410 ggg cca gat ggt aag ccc tcc cgt atc gta gtt atc tac acg acg ggg 3440Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly 415 420 425 430 agt cag gca act atg gat gaa cga aat aga cag atc gct gag ata ggt 3488Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly 435 440 445 gcc tca ctg att aag cat tgg taa ctgtcagacc aagtttactc atatatactt 3542Ala Ser Leu Ile Lys His Trp 450 tagattgatt tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 3602cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 3662ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 3722gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc 3782acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 3842ctttaatagt ggactcttgt tccaaacttg aacaacactc aaccctatct cgggctattc 3902ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 3962acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttaaa aggatctagg 4022tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 4082gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 4142taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 4202aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 4262ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 4322catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 4382ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 4442ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 4502agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 4562taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 4622atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 4682cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 4742ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 4802accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 4862gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 4922tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 4982agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca 5042cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 5102acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 5162acgcgcgagg cagcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca 5222tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg 5282tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga 5342tgcgtccggc gtagaggatc tgctcatgtt tgacagctta tc 5384883PRTEscherichia coli 8Met Arg Thr Ile Ser Tyr Ser Glu Ala Arg Gln Asn Leu Ser Ala Thr 1 5 10 15 Met Met Lys Ala Val Glu Asp His Ala Pro Ile Leu Ile Thr Arg Gln 20 25 30 Asn Gly Glu Ala Cys Val Leu Met Ser Leu Glu Glu Tyr Asn Ser Leu 35 40 45 Glu Glu Thr Ala Tyr Leu Leu Arg Ser Pro Ala Asn Ala Arg Arg Leu 50 55 60 Met Asp Ser Ile Asp Ser Leu Lys Ser Gly Lys Gly Thr Glu Lys Asp 65 70 75 80 Ile Ile Glu 984PRTEscherichia coli 9Met Lys Leu Ile Trp Ser Glu Glu Ser Trp Asp Asp Tyr Leu Tyr Trp 1 5 10 15 Gln Glu Thr Asp Lys Arg Ile Val Lys Lys Ile Asn Glu Leu Ile Lys 20 25 30 Asp Thr Arg Arg Thr Pro Phe Glu Gly Lys Gly Lys Pro Glu Pro Leu 35 40 45 Lys His Asn Leu Ser Gly Phe Trp Ser Arg Arg Ile Thr Glu Glu His 50 55 60 Arg Leu Val Tyr Ala Val Thr Asp Asp Ser Leu Leu Ile Ala Ala Cys 65 70 75 80 Arg Tyr His Tyr 10286PRTEscherichia coli 10Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30 Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp 35 40 45 Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 50 55 60 Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 65 70 75 80 Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 85 90 95 Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 100 105 110 Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser 115 120 125 Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys 130 135 140 Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu 145 150 155 160 Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg 165 170 175 Asp Thr Thr Met Pro Ala Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 180 185 190 Thr Gly

Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp 195 200 205 Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 210 215 220 Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser 225 230 235 240 Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile 245 250 255 Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn 260 265 270 Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp 275 280 285 11879DNAEscherichia coliCDS(1)..(879)araCmev mevalonate sensor protein 11atg gct gaa gcg caa aat gat ccc ctg ctg ccg gga tac tcg ttt aat 48Met Ala Glu Ala Gln Asn Asp Pro Leu Leu Pro Gly Tyr Ser Phe Asn 1 5 10 15 gcc cat ctg gtg gcg ggt tta ctc ccg att gag gcc aac ggt tat ctc 96Ala His Leu Val Ala Gly Leu Leu Pro Ile Glu Ala Asn Gly Tyr Leu 20 25 30 gat ttt ttt atc gac cga ccg ctg gga atg aaa ggt tat att ctc aat 144Asp Phe Phe Ile Asp Arg Pro Leu Gly Met Lys Gly Tyr Ile Leu Asn 35 40 45 ctc acc att cgc ggt cag ggg gtg gtg aaa aat cag gga cga gaa ttt 192Leu Thr Ile Arg Gly Gln Gly Val Val Lys Asn Gln Gly Arg Glu Phe 50 55 60 gtt tgc cga ccg ggt gat att ttg ctg ttc ccg cca gga gag att ctg 240Val Cys Arg Pro Gly Asp Ile Leu Leu Phe Pro Pro Gly Glu Ile Leu 65 70 75 80 cac ttg ggt cgt cat ccg gag gct cgc gaa tgg tat cga cag tgg gtt 288His Leu Gly Arg His Pro Glu Ala Arg Glu Trp Tyr Arg Gln Trp Val 85 90 95 tac ttt cgt ccg cgc gcc tac tgg cat gaa tgg ctt aac tgg ccg tca 336Tyr Phe Arg Pro Arg Ala Tyr Trp His Glu Trp Leu Asn Trp Pro Ser 100 105 110 ata ttt gcc aat acg ggg ttc ttt cgc ccg gat gaa gcg cac cag ccg 384Ile Phe Ala Asn Thr Gly Phe Phe Arg Pro Asp Glu Ala His Gln Pro 115 120 125 cat ttc agc gac ctg ttt ggg caa atc att aac gcc ggg caa ggg gaa 432His Phe Ser Asp Leu Phe Gly Gln Ile Ile Asn Ala Gly Gln Gly Glu 130 135 140 ggg cgc tat tcg gag ctg ctg gcg ata aat ctg ctt gag caa ttg tta 480Gly Arg Tyr Ser Glu Leu Leu Ala Ile Asn Leu Leu Glu Gln Leu Leu 145 150 155 160 ctg cgg cgc atg gaa gcg att aac gag tcg ctc cat cca ccg atg gat 528Leu Arg Arg Met Glu Ala Ile Asn Glu Ser Leu His Pro Pro Met Asp 165 170 175 aat cgg gta cgc gag gct tgt cag tac atc agc gat cac ctg gca gac 576Asn Arg Val Arg Glu Ala Cys Gln Tyr Ile Ser Asp His Leu Ala Asp 180 185 190 agc aat ttt gat atc gcc agc gtc gca cag cat gtt tgc ttg tcg ccg 624Ser Asn Phe Asp Ile Ala Ser Val Ala Gln His Val Cys Leu Ser Pro 195 200 205 tcg cgt ctg tca cat ctt ttc cgc cag cag tta ggg att agc gtc tta 672Ser Arg Leu Ser His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val Leu 210 215 220 agc tgg cgc gag gac caa cgt atc agc cag gcg aag ctg ctt ttg agc 720Ser Trp Arg Glu Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu Ser 225 230 235 240 acc acc cgg atg cct atc gcc acc gtc ggt cgc aat gtt ggt ttt gac 768Thr Thr Arg Met Pro Ile Ala Thr Val Gly Arg Asn Val Gly Phe Asp 245 250 255 gat caa ctc tat ttc tcg cgg gta ttt aaa aaa tgc acc ggg gcc agc 816Asp Gln Leu Tyr Phe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala Ser 260 265 270 ccg agc gag ttc cgt gcc ggt tgt gaa gaa aaa gtg aat gat gta gcc 864Pro Ser Glu Phe Arg Ala Gly Cys Glu Glu Lys Val Asn Asp Val Ala 275 280 285 gtc aag ttg tca taa 879Val Lys Leu Ser 290 12292PRTEscherichia coli 12Met Ala Glu Ala Gln Asn Asp Pro Leu Leu Pro Gly Tyr Ser Phe Asn 1 5 10 15 Ala His Leu Val Ala Gly Leu Leu Pro Ile Glu Ala Asn Gly Tyr Leu 20 25 30 Asp Phe Phe Ile Asp Arg Pro Leu Gly Met Lys Gly Tyr Ile Leu Asn 35 40 45 Leu Thr Ile Arg Gly Gln Gly Val Val Lys Asn Gln Gly Arg Glu Phe 50 55 60 Val Cys Arg Pro Gly Asp Ile Leu Leu Phe Pro Pro Gly Glu Ile Leu 65 70 75 80 His Leu Gly Arg His Pro Glu Ala Arg Glu Trp Tyr Arg Gln Trp Val 85 90 95 Tyr Phe Arg Pro Arg Ala Tyr Trp His Glu Trp Leu Asn Trp Pro Ser 100 105 110 Ile Phe Ala Asn Thr Gly Phe Phe Arg Pro Asp Glu Ala His Gln Pro 115 120 125 His Phe Ser Asp Leu Phe Gly Gln Ile Ile Asn Ala Gly Gln Gly Glu 130 135 140 Gly Arg Tyr Ser Glu Leu Leu Ala Ile Asn Leu Leu Glu Gln Leu Leu 145 150 155 160 Leu Arg Arg Met Glu Ala Ile Asn Glu Ser Leu His Pro Pro Met Asp 165 170 175 Asn Arg Val Arg Glu Ala Cys Gln Tyr Ile Ser Asp His Leu Ala Asp 180 185 190 Ser Asn Phe Asp Ile Ala Ser Val Ala Gln His Val Cys Leu Ser Pro 195 200 205 Ser Arg Leu Ser His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val Leu 210 215 220 Ser Trp Arg Glu Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu Ser 225 230 235 240 Thr Thr Arg Met Pro Ile Ala Thr Val Gly Arg Asn Val Gly Phe Asp 245 250 255 Asp Gln Leu Tyr Phe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala Ser 260 265 270 Pro Ser Glu Phe Arg Ala Gly Cys Glu Glu Lys Val Asn Asp Val Ala 275 280 285 Val Lys Leu Ser 290 131772DNAEscherichia coliCDS(649)..(1443)Neomycin phosphotransferase II protein 13cgttatctcc tgatacacct tagatctata aggctacgct agcgtatcaa aactgacaat 60tcattctatg aatgaatctg ttcaataatg ataacgacat gctgcaatga gcagtgaatc 120gtcggtaacc gcgtatacca gacggtgctc ctctgtaatg cgtcgggacc agaaacctga 180caaattatgt ttcaggggtt ctggcttcca catatgaata ttccggggat ccgtcgacct 240gcagttcgaa gttcctattc tctagaaagt ataggaactt cagagcgctt ttgaagctca 300cgctgccgca agcactcagg gcgcaagggc tgctaaagga agcggaacac gtagaaagcc 360agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat ctggacaagg 420gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta 480gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt 540aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg 600cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgc atg att gaa 657 Met Ile Glu 1 caa gat gga ttg cac gca ggt tct ccg gcc gct tgg gtg gag agg cta 705Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val Glu Arg Leu 5 10 15 ttc ggc tat gac tgg gca caa cag aca atc ggc tgc tct gat gcc gcc 753Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser Asp Ala Ala 20 25 30 35 gtg ttc cgg ctg tca gcg cag ggg cgc ccg gtt ctt ttt gtc aag acc 801Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe Val Lys Thr 40 45 50 gac ctg tcc ggt gcc ctg aat gaa ctg cag gac gag gca gcg cgg cta 849Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala Ala Arg Leu 55 60 65 tcg tgg ctg gcc acg acg ggc gtt cct tgc gca gct gtg ctc gac gtt 897Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val Leu Asp Val 70 75 80 gtc act gaa gcg gga agg gac tgg ctg cta ttg ggc gaa gtg ccg ggg 945Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu Val Pro Gly 85 90 95 cag gat ctc ctg tca tct cac ctt gct cct gcc gag aaa gta tcc atc 993Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys Val Ser Ile 100 105 110 115 atg gct gat gca atg cgg cgg ctg cat acg ctt gat ccg gct acc tgc 1041Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro Ala Thr Cys 120 125 130 cca ttc gac cac caa gcg aaa cat cgc atc gag cga gca cgt act cgg 1089Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg 135 140 145 atg gaa gcc ggt ctt gtc gat cag gat gat ctg gac gaa gag cat cag 1137Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu Glu His Gln 150 155 160 ggg ctc gcg cca gcc gaa ctg ttc gcc agg ctc aag gcg cgc atg ccc 1185Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala Arg Met Pro 165 170 175 gac ggc gag gat ctc gtc gtg acc cat ggc gat gcc tgc ttg ccg aat 1233Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys Leu Pro Asn 180 185 190 195 atc atg gtg gaa aat ggc cgc ttt tct gga ttc atc gac tgt ggc cgg 1281Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg 200 205 210 ctg ggt gtg gcg gac cgc tat cag gac ata gcg ttg gct acc cgt gat 1329Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala Thr Arg Asp 215 220 225 att gct gaa gag ctt ggc ggc gaa tgg gct gac cgc ttc ctc gtg ctt 1377Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe Leu Val Leu 230 235 240 tac ggt atc gcc gct ccc gat tcg cag cgc atc gcc ttc tat cgc ctt 1425Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe Tyr Arg Leu 245 250 255 ctt gac gag ttc ttc taa taaggggatc ttgaagttcc tattccgaag 1473Leu Asp Glu Phe Phe 260 ttcctattct ctagaaagta taggaacttc gaagcagctc cagcctacac aatcgctgtc 1533aatctcctct tttgtacagt tcattgtaca atgatgagcg ttaattaact atttattaat 1593tagtttgtag atcaaggtat tgtcagtgag acgaaaatcc aggcttcgct atttttggtg 1653ccatcagcta agaggacagt cctcttagcc ccctcctttc cccgctcatt cattaaacaa 1713atccattgcc ataaaatata taaaaaagcc cttgctttct aacgtgaaag tggtttagg 177214264PRTEscherichia coli 14Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val 1 5 10 15 Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser 20 25 30 Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe 35 40 45 Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala 50 55 60 Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val 65 70 75 80 Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu 85 90 95 Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys 100 105 110 Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro 115 120 125 Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala 130 135 140 Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu 145 150 155 160 Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala 165 170 175 Arg Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys 180 185 190 Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp 195 200 205 Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala 210 215 220 Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe 225 230 235 240 Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe 245 250 255 Tyr Arg Leu Leu Asp Glu Phe Phe 260 156707DNAEscherichia colipromoter(149)..(181)J23100 promoterCDS(255)..(1439)E. coli atoB gene encoding acetyl-CoA acetyltransferaseCDS(1461)..(2627)L. casei mvaS gene encoding HMG-CoA synthaseCDS(2648)..(3928)L. casei mvaE gene encoding HMG-CoA reductaserep_origin(5766)..(6605)p15A origin of replication 15acgatgcgtc cggcgtagag gatctgctca tgtttgacag cttatcatcg atgcatgcgc 60ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac 120aggtttcccg actggaaagc gactcattga cggctagctc agtcctaggt acagtgctag 180cattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgaa ttcattaaag 240aggagaaagg tacc atg aaa aat tgt gtc atc gtc agt gcg gta cgt act 290 Met Lys Asn Cys Val Ile Val Ser Ala Val Arg Thr 1 5 10 gct atc ggt agt ttt aac ggt tca ctc gct tcc acc agc gcc atc gac 338Ala Ile Gly Ser Phe Asn Gly Ser Leu Ala Ser Thr Ser Ala Ile Asp 15 20 25 ctg ggg gcg aca gta att aaa gcc gcc att gaa cgt gca aaa atc gat 386Leu Gly Ala Thr Val Ile Lys Ala Ala Ile Glu Arg Ala Lys Ile Asp 30 35 40 tca caa cac gtt gat gaa gtg att atg ggt aac gtg tta caa gcc ggg 434Ser Gln His Val Asp Glu Val Ile Met Gly Asn Val Leu Gln Ala Gly 45 50 55 60 ctg ggg caa aat ccg gcg cgt cag gca ctg tta aaa agc ggg ctg gca 482Leu Gly Gln Asn Pro Ala Arg Gln Ala Leu Leu Lys Ser Gly Leu Ala 65 70 75 gaa acg gtg tgc gga ttc acg gtc aat aaa gta tgt ggt tcg ggt ctt 530Glu Thr Val Cys Gly Phe Thr Val Asn Lys Val Cys Gly Ser Gly Leu 80 85 90 aaa agt gtg gcg ctt gcc gcc cag gcc att cag gca ggt cag gcg cag 578Lys Ser Val Ala Leu Ala Ala Gln Ala Ile Gln Ala Gly Gln Ala Gln 95 100 105 agc att gtg gcg ggg ggt atg gaa aat atg agt tta gcc ccc tac tta 626Ser Ile Val Ala Gly Gly Met Glu Asn Met Ser Leu Ala Pro Tyr Leu 110 115 120 ctc gat gca aaa gca cgc tct ggt tat cgt ctt gga gac gga cag gtt 674Leu Asp Ala Lys Ala Arg Ser Gly Tyr Arg Leu Gly Asp Gly Gln Val 125 130 135 140 tat gac gta atc ctg cgc gat ggc ctg atg tgc gcc acc cat ggt tat 722Tyr Asp Val Ile Leu Arg Asp Gly Leu Met Cys Ala Thr His Gly Tyr 145 150 155 cat atg ggg att acc gcc gaa aac gtg gct aaa gag tac gga att acc 770His Met Gly Ile Thr Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr 160 165 170 cgt gaa atg cag gat gaa ctg gcg cta cat tca cag cgt aaa gcg gca 818Arg Glu Met Gln Asp Glu Leu Ala Leu His Ser Gln Arg Lys Ala Ala 175 180 185 gcc gca att gag tcc ggt gct ttt aca gcc gaa atc gtc ccg gta aat 866Ala Ala Ile Glu Ser Gly Ala Phe Thr Ala Glu Ile Val Pro Val Asn 190 195 200 gtt gtc act cga aag aaa acc ttc gtc ttc agt caa gac gaa ttc ccg 914Val Val Thr Arg Lys Lys Thr Phe Val Phe Ser Gln Asp Glu Phe Pro 205 210 215 220 aaa gcg aat tca acg gct gaa gcg tta ggt gca ttg cgc ccg gcc ttc 962Lys Ala Asn Ser Thr Ala Glu Ala Leu Gly Ala Leu Arg Pro Ala Phe

225 230 235 gat aaa gca gga aca gtc acc gct ggg aac gcg tct ggt att aac gac 1010Asp Lys Ala Gly Thr Val Thr Ala Gly Asn Ala Ser Gly Ile Asn Asp 240 245 250 ggt gct gcc gct ctg gtg att atg gaa gaa tct gcg gcg ctg gca gca 1058Gly Ala Ala Ala Leu Val Ile Met Glu Glu Ser Ala Ala Leu Ala Ala 255 260 265 ggc ctt acc ccc ctg gct cgc att aaa agt tat gcc agc ggt ggc gtg 1106Gly Leu Thr Pro Leu Ala Arg Ile Lys Ser Tyr Ala Ser Gly Gly Val 270 275 280 ccc ccc gca ttg atg ggt atg ggg cca gta cct gcc acg caa aaa gcg 1154Pro Pro Ala Leu Met Gly Met Gly Pro Val Pro Ala Thr Gln Lys Ala 285 290 295 300 tta caa ctg gcg ggg ctg caa ctg gcg gat att gat ctc att gag gct 1202Leu Gln Leu Ala Gly Leu Gln Leu Ala Asp Ile Asp Leu Ile Glu Ala 305 310 315 aat gaa gca ttt gct gca cag ttc ctt gcc gtt ggg aaa aac ctg ggc 1250Asn Glu Ala Phe Ala Ala Gln Phe Leu Ala Val Gly Lys Asn Leu Gly 320 325 330 ttt gat tct gag aaa gtg aat gtc aac ggc ggg gcc atc gcg ctc ggg 1298Phe Asp Ser Glu Lys Val Asn Val Asn Gly Gly Ala Ile Ala Leu Gly 335 340 345 cat cct atc ggt gcc agt ggt gct cgt att ctg gtc aca cta tta cat 1346His Pro Ile Gly Ala Ser Gly Ala Arg Ile Leu Val Thr Leu Leu His 350 355 360 gcc atg cag gca cgc gat aaa acg ctg ggg ctg gca aca ctg tgc att 1394Ala Met Gln Ala Arg Asp Lys Thr Leu Gly Leu Ala Thr Leu Cys Ile 365 370 375 380 ggc ggc ggt cag gga att gcg atg gtg att gaa cgg ttg aat taa 1439Gly Gly Gly Gln Gly Ile Ala Met Val Ile Glu Arg Leu Asn 385 390 gctgcagagg agaaattaac t atg aaa atc ggg att gat gca atc gct atg 1490 Met Lys Ile Gly Ile Asp Ala Ile Ala Met 395 400 gac acc cca gat ttc tac gtt gat tta gta aaa ctc gcg cag gtt cga 1538Asp Thr Pro Asp Phe Tyr Val Asp Leu Val Lys Leu Ala Gln Val Arg 405 410 415 420 ggt gat gat ccg gac aaa tat acg att ggc att ggc caa gac gag caa 1586Gly Asp Asp Pro Asp Lys Tyr Thr Ile Gly Ile Gly Gln Asp Glu Gln 425 430 435 gcg gta cca cct tct agc caa gac att gtc acg atg ggt gcg aat gca 1634Ala Val Pro Pro Ser Ser Gln Asp Ile Val Thr Met Gly Ala Asn Ala 440 445 450 gcg aca aag ttg ttg acg cca gcg att cgc gct agc ttg ggc atg gtc 1682Ala Thr Lys Leu Leu Thr Pro Ala Ile Arg Ala Ser Leu Gly Met Val 455 460 465 ttg gtt ggt act gaa agt ggt gtt gat gcc agt aaa tcg gct gca ttg 1730Leu Val Gly Thr Glu Ser Gly Val Asp Ala Ser Lys Ser Ala Ala Leu 470 475 480 ttt att cat gat tta ttg gca ctc cct gag tgg gtg cgg gcc gtt gag 1778Phe Ile His Asp Leu Leu Ala Leu Pro Glu Trp Val Arg Ala Val Glu 485 490 495 500 tta aag gaa gct tgt tat ggc ggt act gcc gca cta atg atg gcg cgt 1826Leu Lys Glu Ala Cys Tyr Gly Gly Thr Ala Ala Leu Met Met Ala Arg 505 510 515 gac tac atc gcc gct cac cca gat aaa acc gtt tta gtc att gct gcg 1874Asp Tyr Ile Ala Ala His Pro Asp Lys Thr Val Leu Val Ile Ala Ala 520 525 530 gat atc gcc cgg tac ggt ttg gca aca gca gga gaa gtc aca caa ggt 1922Asp Ile Ala Arg Tyr Gly Leu Ala Thr Ala Gly Glu Val Thr Gln Gly 535 540 545 gcg ggc gcg gtt gcc atg tta atc aaa gca gag ccg cac atc atg acc 1970Ala Gly Ala Val Ala Met Leu Ile Lys Ala Glu Pro His Ile Met Thr 550 555 560 att gaa gac gat tcg gtt tat cgg tct gaa tct att gac gat ttt tgg 2018Ile Glu Asp Asp Ser Val Tyr Arg Ser Glu Ser Ile Asp Asp Phe Trp 565 570 575 580 cgg ccg gtt tat caa gac aca gca att gca caa gga aag tat tca acg 2066Arg Pro Val Tyr Gln Asp Thr Ala Ile Ala Gln Gly Lys Tyr Ser Thr 585 590 595 gaa caa tat tta gct ttt ttt caa gca att tgg tca cga tac cag acc 2114Glu Gln Tyr Leu Ala Phe Phe Gln Ala Ile Trp Ser Arg Tyr Gln Thr 600 605 610 caa cgc cat cac aca gca agt gat ttt gct gcg atg aca ttc cat tta 2162Gln Arg His His Thr Ala Ser Asp Phe Ala Ala Met Thr Phe His Leu 615 620 625 ccg tat acc aaa atg ggc aag aaa gct tta aag ctt gtt ctg ccg gac 2210Pro Tyr Thr Lys Met Gly Lys Lys Ala Leu Lys Leu Val Leu Pro Asp 630 635 640 aca gat gaa gcc act ggc gag cga ctg caa cgg cgc ttt gag gca agc 2258Thr Asp Glu Ala Thr Gly Glu Arg Leu Gln Arg Arg Phe Glu Ala Ser 645 650 655 660 act cgg tac tgc cgg cga gtt ggc aat att tat acc ggt tct ttg tat 2306Thr Arg Tyr Cys Arg Arg Val Gly Asn Ile Tyr Thr Gly Ser Leu Tyr 665 670 675 ttg ggt ctg ctg tcg ttg ttg gac aat gat act agc ttg aaa gcg cgt 2354Leu Gly Leu Leu Ser Leu Leu Asp Asn Asp Thr Ser Leu Lys Ala Arg 680 685 690 gac cgg atc ggt tta ttc tcg tat ggc tcc ggg gcc gtg gca gaa ttt 2402Asp Arg Ile Gly Leu Phe Ser Tyr Gly Ser Gly Ala Val Ala Glu Phe 695 700 705 ttt agc ggc atc ttg caa ccg gat ttt gcc gca caa ctg cat gca gcc 2450Phe Ser Gly Ile Leu Gln Pro Asp Phe Ala Ala Gln Leu His Ala Ala 710 715 720 aat cac gct aaa atg ttg gct gat cgt cag gaa tta acc gtt cct gaa 2498Asn His Ala Lys Met Leu Ala Asp Arg Gln Glu Leu Thr Val Pro Glu 725 730 735 740 tac gaa gct gtt ttc agc gat aag gtg cct tat gat cca gaa gat tat 2546Tyr Glu Ala Val Phe Ser Asp Lys Val Pro Tyr Asp Pro Glu Asp Tyr 745 750 755 cgt agt gat ccg act tat tat cat ggt cag ttt gtt ttg acc ggt gtg 2594Arg Ser Asp Pro Thr Tyr Tyr His Gly Gln Phe Val Leu Thr Gly Val 760 765 770 atc ggt caa gaa cgt caa tat cag cag cgg taa ggatccagga gaaattaact 2647Ile Gly Gln Glu Arg Gln Tyr Gln Gln Arg 775 780 atg aaa ttt tac gag ttg tct ccg gaa aaa cgc cgt gac caa ttg gtt 2695Met Lys Phe Tyr Glu Leu Ser Pro Glu Lys Arg Arg Asp Gln Leu Val 785 790 795 caa gaa ggg tgg tta aca aca cag gat gct gcg ttg tta gct ggt acg 2743Gln Glu Gly Trp Leu Thr Thr Gln Asp Ala Ala Leu Leu Ala Gly Thr 800 805 810 cat agc ttg cct gaa gtc aca gga gcg cgt ctg atc gaa aat gcc atc 2791His Ser Leu Pro Glu Val Thr Gly Ala Arg Leu Ile Glu Asn Ala Ile 815 820 825 830 ggc gag ttc cct tta ccc cta ggc gtt gct cgc aat tta ttg gtc aat 2839Gly Glu Phe Pro Leu Pro Leu Gly Val Ala Arg Asn Leu Leu Val Asn 835 840 845 gga cag cta cat caa gtg cct ata gcg gat gag gaa cct tct gtg att 2887Gly Gln Leu His Gln Val Pro Ile Ala Asp Glu Glu Pro Ser Val Ile 850 855 860 gca gct gcc agt aat ggg gcc agg ctt gcg act gca aac ggg ggc gtt 2935Ala Ala Ala Ser Asn Gly Ala Arg Leu Ala Thr Ala Asn Gly Gly Val 865 870 875 agg acc cat gta gcg gcg cat cga gtg gtc gct gaa gta gtc ctg act 2983Arg Thr His Val Ala Ala His Arg Val Val Ala Glu Val Val Leu Thr 880 885 890 aac ttg acg gac tta gtg caa gca agg caa acg att ttg gct cat cag 3031Asn Leu Thr Asp Leu Val Gln Ala Arg Gln Thr Ile Leu Ala His Gln 895 900 905 910 act gat att cag aaa gtc att gcg gtt gcg cat cct tcg atg att caa 3079Thr Asp Ile Gln Lys Val Ile Ala Val Ala His Pro Ser Met Ile Gln 915 920 925 cgt ggc ggt ggt ctt gat cag tta acg gtc gaa tca cta gga gca cag 3127Arg Gly Gly Gly Leu Asp Gln Leu Thr Val Glu Ser Leu Gly Ala Gln 930 935 940 ttc ttg aaa atc cgt tta acg ctc gat ccg caa cag gca atg ggg gcc 3175Phe Leu Lys Ile Arg Leu Thr Leu Asp Pro Gln Gln Ala Met Gly Ala 945 950 955 aat tat gcg aat aca gtt gcc gaa gcg gtc gca gcg gcg gtg aca agc 3223Asn Tyr Ala Asn Thr Val Ala Glu Ala Val Ala Ala Ala Val Thr Ser 960 965 970 tgg gta gac ggt gat gtg ctt gtc agt att tta act aac gcg cca aca 3271Trp Val Asp Gly Asp Val Leu Val Ser Ile Leu Thr Asn Ala Pro Thr 975 980 985 990 gaa ctc gtg acg gct gag gtt tca ctt gag ccg gtt tct tta gcg acg 3319Glu Leu Val Thr Ala Glu Val Ser Leu Glu Pro Val Ser Leu Ala Thr 995 1000 1005 aaa gct ctt tct gga gat gtg att gct aag aaa att gtt cag ctt 3364Lys Ala Leu Ser Gly Asp Val Ile Ala Lys Lys Ile Val Gln Leu 1010 1015 1020 agc gat tta gcc ttt gtc gat gct gag cgg gca gtg acc cac aac 3409Ser Asp Leu Ala Phe Val Asp Ala Glu Arg Ala Val Thr His Asn 1025 1030 1035 aag ggc att ctt aac ggt att att ggt gct gta ttg gct act ggc 3454Lys Gly Ile Leu Asn Gly Ile Ile Gly Ala Val Leu Ala Thr Gly 1040 1045 1050 aat gat acc cgc gct gtc gca gca agt atc ggt gca ttt gca tgt 3499Asn Asp Thr Arg Ala Val Ala Ala Ser Ile Gly Ala Phe Ala Cys 1055 1060 1065 gcg tct ggc agg tat cag cct tta tcg cgc tgg tat atg gat cag 3544Ala Ser Gly Arg Tyr Gln Pro Leu Ser Arg Trp Tyr Met Asp Gln 1070 1075 1080 ggc cat tta gtt ggt cac tta cag ctg ccc ttg ccg atg ggg gca 3589Gly His Leu Val Gly His Leu Gln Leu Pro Leu Pro Met Gly Ala 1085 1090 1095 gtt ggc ggg gcg atc ggt gct tta cca atg gcg cag gtt gtc cgt 3634Val Gly Gly Ala Ile Gly Ala Leu Pro Met Ala Gln Val Val Arg 1100 1105 1110 cgg ctc ggt ggg tat cag aat tta gct atc atg cag caa gtt att 3679Arg Leu Gly Gly Tyr Gln Asn Leu Ala Ile Met Gln Gln Val Ile 1115 1120 1125 gcc gcc ctc ggg ttg gtg caa aat ctt gct gcg atg cga gca ttg 3724Ala Ala Leu Gly Leu Val Gln Asn Leu Ala Ala Met Arg Ala Leu 1130 1135 1140 gct gga ccg ggg att caa gct ggt cac atg aag ttg cag gct aat 3769Ala Gly Pro Gly Ile Gln Ala Gly His Met Lys Leu Gln Ala Asn 1145 1150 1155 gcc ctc gcc att gct gcc ggg gca aca gaa aca gaa ttg ccg atg 3814Ala Leu Ala Ile Ala Ala Gly Ala Thr Glu Thr Glu Leu Pro Met 1160 1165 1170 ctt gtg aac gca ctt cgt caa ggt agt atg gat tta aaa cat gcg 3859Leu Val Asn Ala Leu Arg Gln Gly Ser Met Asp Leu Lys His Ala 1175 1180 1185 caa caa tat tta aca acc att cgt tta aac aag aaa gta ggc caa 3904Gln Gln Tyr Leu Thr Thr Ile Arg Leu Asn Lys Lys Val Gly Gln 1190 1195 1200 tca aaa gat gaa aat cgg gat tga gtcgacctgc aggcatgcaa gcttggctgt 3958Ser Lys Asp Glu Asn Arg Asp 1205 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt 4018ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg 4078aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta 4138gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 4198tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt 4258gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag 4318gcatcaaatt aagcagaagg ccatcctgac ggtaccgcta ccagcggtgg tttgtttgcc 4378ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 4438aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 4498gcctacatac ctcgctctgc taatcctgtt accagtgggg catttgagaa gcacacggtc 4558acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa 4618cgaccctgcc ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg ccattcatcc 4678gcttattatc acttattcag gcgtagcacc aggcgtttaa gggcaccaat aactgcctta 4738aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct 4798gccgacatgg aagccatcac agacggcatg atgaacctga atcgccagcg gcatcagcac 4858cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat 4918attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa 4978catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc 5038ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga 5098aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac 5158cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag 5218aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc 5278cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc 5338aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt 5398ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag 5458tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt 5518tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc 5578tgcgaagtga tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc 5638caacttactg atttagtgta tgatggtgtt tttgaggtgc tccagtggct tctgtttcta 5698tcagctgtcc ctcctgttca gctactgacg gggtggtgcg taacggcaaa agcaccgccg 5758gacatcagcg ctagcggagt gtatactggc ttactatgtt ggcactgatg agggtgtcag 5818tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga 5878tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg 5938cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt 5998aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca 6058agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat 6118accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta 6178ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc 6238gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc 6298tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca 6358ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta 6418aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt 6478caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt 6538ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca 6598gataaaatat ttgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc 6658ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcc 670716394PRTEscherichia coli 16Met Lys Asn Cys Val Ile Val Ser Ala Val Arg Thr Ala Ile Gly Ser 1 5 10 15 Phe Asn Gly Ser Leu Ala Ser Thr Ser Ala Ile Asp Leu Gly Ala Thr 20 25 30 Val Ile Lys Ala Ala Ile Glu Arg Ala Lys Ile Asp Ser Gln His Val 35 40 45 Asp Glu Val Ile Met Gly Asn Val Leu Gln Ala Gly Leu Gly Gln Asn 50 55 60 Pro Ala Arg Gln Ala Leu Leu Lys Ser Gly Leu Ala Glu Thr Val Cys 65 70 75 80 Gly Phe Thr Val Asn Lys Val Cys Gly Ser Gly Leu Lys Ser Val Ala 85 90 95 Leu Ala Ala Gln Ala Ile Gln Ala Gly Gln Ala Gln Ser Ile Val Ala 100 105 110 Gly Gly Met Glu Asn Met Ser Leu Ala Pro Tyr Leu Leu Asp Ala Lys 115 120 125 Ala Arg Ser Gly Tyr Arg Leu Gly Asp Gly Gln Val Tyr Asp Val Ile 130 135 140

Leu Arg Asp Gly Leu Met Cys Ala Thr His Gly Tyr His Met Gly Ile 145 150 155 160 Thr Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu Met Gln 165 170 175 Asp Glu Leu Ala Leu His Ser Gln Arg Lys Ala Ala Ala Ala Ile Glu 180 185 190 Ser Gly Ala Phe Thr Ala Glu Ile Val Pro Val Asn Val Val Thr Arg 195 200 205 Lys Lys Thr Phe Val Phe Ser Gln Asp Glu Phe Pro Lys Ala Asn Ser 210 215 220 Thr Ala Glu Ala Leu Gly Ala Leu Arg Pro Ala Phe Asp Lys Ala Gly 225 230 235 240 Thr Val Thr Ala Gly Asn Ala Ser Gly Ile Asn Asp Gly Ala Ala Ala 245 250 255 Leu Val Ile Met Glu Glu Ser Ala Ala Leu Ala Ala Gly Leu Thr Pro 260 265 270 Leu Ala Arg Ile Lys Ser Tyr Ala Ser Gly Gly Val Pro Pro Ala Leu 275 280 285 Met Gly Met Gly Pro Val Pro Ala Thr Gln Lys Ala Leu Gln Leu Ala 290 295 300 Gly Leu Gln Leu Ala Asp Ile Asp Leu Ile Glu Ala Asn Glu Ala Phe 305 310 315 320 Ala Ala Gln Phe Leu Ala Val Gly Lys Asn Leu Gly Phe Asp Ser Glu 325 330 335 Lys Val Asn Val Asn Gly Gly Ala Ile Ala Leu Gly His Pro Ile Gly 340 345 350 Ala Ser Gly Ala Arg Ile Leu Val Thr Leu Leu His Ala Met Gln Ala 355 360 365 Arg Asp Lys Thr Leu Gly Leu Ala Thr Leu Cys Ile Gly Gly Gly Gln 370 375 380 Gly Ile Ala Met Val Ile Glu Arg Leu Asn 385 390 17388PRTEscherichia coli 17Met Lys Ile Gly Ile Asp Ala Ile Ala Met Asp Thr Pro Asp Phe Tyr 1 5 10 15 Val Asp Leu Val Lys Leu Ala Gln Val Arg Gly Asp Asp Pro Asp Lys 20 25 30 Tyr Thr Ile Gly Ile Gly Gln Asp Glu Gln Ala Val Pro Pro Ser Ser 35 40 45 Gln Asp Ile Val Thr Met Gly Ala Asn Ala Ala Thr Lys Leu Leu Thr 50 55 60 Pro Ala Ile Arg Ala Ser Leu Gly Met Val Leu Val Gly Thr Glu Ser 65 70 75 80 Gly Val Asp Ala Ser Lys Ser Ala Ala Leu Phe Ile His Asp Leu Leu 85 90 95 Ala Leu Pro Glu Trp Val Arg Ala Val Glu Leu Lys Glu Ala Cys Tyr 100 105 110 Gly Gly Thr Ala Ala Leu Met Met Ala Arg Asp Tyr Ile Ala Ala His 115 120 125 Pro Asp Lys Thr Val Leu Val Ile Ala Ala Asp Ile Ala Arg Tyr Gly 130 135 140 Leu Ala Thr Ala Gly Glu Val Thr Gln Gly Ala Gly Ala Val Ala Met 145 150 155 160 Leu Ile Lys Ala Glu Pro His Ile Met Thr Ile Glu Asp Asp Ser Val 165 170 175 Tyr Arg Ser Glu Ser Ile Asp Asp Phe Trp Arg Pro Val Tyr Gln Asp 180 185 190 Thr Ala Ile Ala Gln Gly Lys Tyr Ser Thr Glu Gln Tyr Leu Ala Phe 195 200 205 Phe Gln Ala Ile Trp Ser Arg Tyr Gln Thr Gln Arg His His Thr Ala 210 215 220 Ser Asp Phe Ala Ala Met Thr Phe His Leu Pro Tyr Thr Lys Met Gly 225 230 235 240 Lys Lys Ala Leu Lys Leu Val Leu Pro Asp Thr Asp Glu Ala Thr Gly 245 250 255 Glu Arg Leu Gln Arg Arg Phe Glu Ala Ser Thr Arg Tyr Cys Arg Arg 260 265 270 Val Gly Asn Ile Tyr Thr Gly Ser Leu Tyr Leu Gly Leu Leu Ser Leu 275 280 285 Leu Asp Asn Asp Thr Ser Leu Lys Ala Arg Asp Arg Ile Gly Leu Phe 290 295 300 Ser Tyr Gly Ser Gly Ala Val Ala Glu Phe Phe Ser Gly Ile Leu Gln 305 310 315 320 Pro Asp Phe Ala Ala Gln Leu His Ala Ala Asn His Ala Lys Met Leu 325 330 335 Ala Asp Arg Gln Glu Leu Thr Val Pro Glu Tyr Glu Ala Val Phe Ser 340 345 350 Asp Lys Val Pro Tyr Asp Pro Glu Asp Tyr Arg Ser Asp Pro Thr Tyr 355 360 365 Tyr His Gly Gln Phe Val Leu Thr Gly Val Ile Gly Gln Glu Arg Gln 370 375 380 Tyr Gln Gln Arg 385 18426PRTEscherichia coli 18Met Lys Phe Tyr Glu Leu Ser Pro Glu Lys Arg Arg Asp Gln Leu Val 1 5 10 15 Gln Glu Gly Trp Leu Thr Thr Gln Asp Ala Ala Leu Leu Ala Gly Thr 20 25 30 His Ser Leu Pro Glu Val Thr Gly Ala Arg Leu Ile Glu Asn Ala Ile 35 40 45 Gly Glu Phe Pro Leu Pro Leu Gly Val Ala Arg Asn Leu Leu Val Asn 50 55 60 Gly Gln Leu His Gln Val Pro Ile Ala Asp Glu Glu Pro Ser Val Ile 65 70 75 80 Ala Ala Ala Ser Asn Gly Ala Arg Leu Ala Thr Ala Asn Gly Gly Val 85 90 95 Arg Thr His Val Ala Ala His Arg Val Val Ala Glu Val Val Leu Thr 100 105 110 Asn Leu Thr Asp Leu Val Gln Ala Arg Gln Thr Ile Leu Ala His Gln 115 120 125 Thr Asp Ile Gln Lys Val Ile Ala Val Ala His Pro Ser Met Ile Gln 130 135 140 Arg Gly Gly Gly Leu Asp Gln Leu Thr Val Glu Ser Leu Gly Ala Gln 145 150 155 160 Phe Leu Lys Ile Arg Leu Thr Leu Asp Pro Gln Gln Ala Met Gly Ala 165 170 175 Asn Tyr Ala Asn Thr Val Ala Glu Ala Val Ala Ala Ala Val Thr Ser 180 185 190 Trp Val Asp Gly Asp Val Leu Val Ser Ile Leu Thr Asn Ala Pro Thr 195 200 205 Glu Leu Val Thr Ala Glu Val Ser Leu Glu Pro Val Ser Leu Ala Thr 210 215 220 Lys Ala Leu Ser Gly Asp Val Ile Ala Lys Lys Ile Val Gln Leu Ser 225 230 235 240 Asp Leu Ala Phe Val Asp Ala Glu Arg Ala Val Thr His Asn Lys Gly 245 250 255 Ile Leu Asn Gly Ile Ile Gly Ala Val Leu Ala Thr Gly Asn Asp Thr 260 265 270 Arg Ala Val Ala Ala Ser Ile Gly Ala Phe Ala Cys Ala Ser Gly Arg 275 280 285 Tyr Gln Pro Leu Ser Arg Trp Tyr Met Asp Gln Gly His Leu Val Gly 290 295 300 His Leu Gln Leu Pro Leu Pro Met Gly Ala Val Gly Gly Ala Ile Gly 305 310 315 320 Ala Leu Pro Met Ala Gln Val Val Arg Arg Leu Gly Gly Tyr Gln Asn 325 330 335 Leu Ala Ile Met Gln Gln Val Ile Ala Ala Leu Gly Leu Val Gln Asn 340 345 350 Leu Ala Ala Met Arg Ala Leu Ala Gly Pro Gly Ile Gln Ala Gly His 355 360 365 Met Lys Leu Gln Ala Asn Ala Leu Ala Ile Ala Ala Gly Ala Thr Glu 370 375 380 Thr Glu Leu Pro Met Leu Val Asn Ala Leu Arg Gln Gly Ser Met Asp 385 390 395 400 Leu Lys His Ala Gln Gln Tyr Leu Thr Thr Ile Arg Leu Asn Lys Lys 405 410 415 Val Gly Gln Ser Lys Asp Glu Asn Arg Asp 420 425 19660DNAEscherichia coliCDS(1)..(660)camR gene encoding chloramphenicol acetyltransferase 19atg gag aaa aaa atc act gga tat acc acc gtt gat ata tcc caa tgg 48Met Glu Lys Lys Ile Thr Gly Tyr Thr Thr Val Asp Ile Ser Gln Trp 1 5 10 15 cat cgt aaa gaa cat ttt gag gca ttt cag tca gtt gct caa tgt acc 96His Arg Lys Glu His Phe Glu Ala Phe Gln Ser Val Ala Gln Cys Thr 20 25 30 tat aac cag acc gtt cag ctg gat att acg gcc ttt tta aag acc gta 144Tyr Asn Gln Thr Val Gln Leu Asp Ile Thr Ala Phe Leu Lys Thr Val 35 40 45 aag aaa aat aag cac aag ttt tat ccg gcc ttt att cac att ctt gcc 192Lys Lys Asn Lys His Lys Phe Tyr Pro Ala Phe Ile His Ile Leu Ala 50 55 60 cgc ctg atg aat gct cat ccg gaa ttc cgt atg gca atg aaa gac ggt 240Arg Leu Met Asn Ala His Pro Glu Phe Arg Met Ala Met Lys Asp Gly 65 70 75 80 gag ctg gtg ata tgg gat agt gtt cac cct tgt tac acc gtt ttc cat 288Glu Leu Val Ile Trp Asp Ser Val His Pro Cys Tyr Thr Val Phe His 85 90 95 gag caa act gaa acg ttt tca tcg ctc tgg agt gaa tac cac gac gat 336Glu Gln Thr Glu Thr Phe Ser Ser Leu Trp Ser Glu Tyr His Asp Asp 100 105 110 ttc cgg cag ttt cta cac ata tat tcg caa gat gtg gcg tgt tac ggt 384Phe Arg Gln Phe Leu His Ile Tyr Ser Gln Asp Val Ala Cys Tyr Gly 115 120 125 gaa aac ctg gcc tat ttc cct aaa ggg ttt att gag aat atg ttt ttc 432Glu Asn Leu Ala Tyr Phe Pro Lys Gly Phe Ile Glu Asn Met Phe Phe 130 135 140 gtc tca gcc aat ccc tgg gtg agt ttc acc agt ttt gat tta aac gtg 480Val Ser Ala Asn Pro Trp Val Ser Phe Thr Ser Phe Asp Leu Asn Val 145 150 155 160 gcc aat atg gac aac ttc ttc gcc ccc gtt ttc acc atg ggc aaa tat 528Ala Asn Met Asp Asn Phe Phe Ala Pro Val Phe Thr Met Gly Lys Tyr 165 170 175 tat acg caa ggc gac aag gtg ctg atg ccg ctg gcg att cag gtt cat 576Tyr Thr Gln Gly Asp Lys Val Leu Met Pro Leu Ala Ile Gln Val His 180 185 190 cat gcc gtc tgt gat ggc ttc cat gtc ggc aga atg ctt aat gaa tta 624His Ala Val Cys Asp Gly Phe His Val Gly Arg Met Leu Asn Glu Leu 195 200 205 caa cag tac tgc gat gag tgg cag ggc ggg gcg taa 660Gln Gln Tyr Cys Asp Glu Trp Gln Gly Gly Ala 210 215 20219PRTEscherichia coli 20Met Glu Lys Lys Ile Thr Gly Tyr Thr Thr Val Asp Ile Ser Gln Trp 1 5 10 15 His Arg Lys Glu His Phe Glu Ala Phe Gln Ser Val Ala Gln Cys Thr 20 25 30 Tyr Asn Gln Thr Val Gln Leu Asp Ile Thr Ala Phe Leu Lys Thr Val 35 40 45 Lys Lys Asn Lys His Lys Phe Tyr Pro Ala Phe Ile His Ile Leu Ala 50 55 60 Arg Leu Met Asn Ala His Pro Glu Phe Arg Met Ala Met Lys Asp Gly 65 70 75 80 Glu Leu Val Ile Trp Asp Ser Val His Pro Cys Tyr Thr Val Phe His 85 90 95 Glu Gln Thr Glu Thr Phe Ser Ser Leu Trp Ser Glu Tyr His Asp Asp 100 105 110 Phe Arg Gln Phe Leu His Ile Tyr Ser Gln Asp Val Ala Cys Tyr Gly 115 120 125 Glu Asn Leu Ala Tyr Phe Pro Lys Gly Phe Ile Glu Asn Met Phe Phe 130 135 140 Val Ser Ala Asn Pro Trp Val Ser Phe Thr Ser Phe Asp Leu Asn Val 145 150 155 160 Ala Asn Met Asp Asn Phe Phe Ala Pro Val Phe Thr Met Gly Lys Tyr 165 170 175 Tyr Thr Gln Gly Asp Lys Val Leu Met Pro Leu Ala Ile Gln Val His 180 185 190 His Ala Val Cys Asp Gly Phe His Val Gly Arg Met Leu Asn Glu Leu 195 200 205 Gln Gln Tyr Cys Asp Glu Trp Gln Gly Gly Ala 210 215 217301DNAEscherichia colipromoter(103)..(135)J23100 promoterCDS(281)..(1465)atoB gene encoding acetyl-CoA acetyltransferaseCDS(1479)..(2954)Mutant ERG13 gene encoding non-functional HMG-CoA synthaseCDS(2968)..(4473)tHMGR gene encoding truncated HMG-CoA reductaserep_origin(6314)..(7153) 21atcgatgcat gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 60gcagctggca cgacaggttt cccgactgga aagcgactca ttgacggcta gctcagtcct 120aggtacagtg ctagcattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc 180tgggtaccgg gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc 240agcccgggga tcctctagag tcgactagga ggaatataaa atg aaa aat tgt gtc 295 Met Lys Asn Cys Val 1 5 atc gtc agt gcg gta cgt act gct atc ggt agt ttt aac ggt tca ctc 343Ile Val Ser Ala Val Arg Thr Ala Ile Gly Ser Phe Asn Gly Ser Leu 10 15 20 gct tcc acc agc gcc atc gac ctg ggg gcg aca gta att aaa gcc gcc 391Ala Ser Thr Ser Ala Ile Asp Leu Gly Ala Thr Val Ile Lys Ala Ala 25 30 35 att gaa cgt gca aaa atc gat tca caa cac gtt gat gaa gtg att atg 439Ile Glu Arg Ala Lys Ile Asp Ser Gln His Val Asp Glu Val Ile Met 40 45 50 ggt aac gtg tta caa gcc ggg ctg ggg caa aat ccg gcg cgt cag gca 487Gly Asn Val Leu Gln Ala Gly Leu Gly Gln Asn Pro Ala Arg Gln Ala 55 60 65 ctg tta aaa agc ggg ctg gca gaa acg gtg tgc gga ttc acg gtc aat 535Leu Leu Lys Ser Gly Leu Ala Glu Thr Val Cys Gly Phe Thr Val Asn 70 75 80 85 aaa gta tgt ggt tcg ggt ctt aaa agt gtg gcg ctt gcc gcc cag gcc 583Lys Val Cys Gly Ser Gly Leu Lys Ser Val Ala Leu Ala Ala Gln Ala 90 95 100 att cag gca ggt cag gcg cag agc att gtg gcg ggg ggt atg gaa aat 631Ile Gln Ala Gly Gln Ala Gln Ser Ile Val Ala Gly Gly Met Glu Asn 105 110 115 atg agt tta gcc ccc tac tta ctc gat gca aaa gca cgc tct ggt tat 679Met Ser Leu Ala Pro Tyr Leu Leu Asp Ala Lys Ala Arg Ser Gly Tyr 120 125 130 cgt ctt gga gac gga cag gtt tat gac gta atc ctg cgc gat ggc ctg 727Arg Leu Gly Asp Gly Gln Val Tyr Asp Val Ile Leu Arg Asp Gly Leu 135 140 145 atg tgc gcc acc cat ggt tat cat atg ggg att acc gcc gaa aac gtg 775Met Cys Ala Thr His Gly Tyr His Met Gly Ile Thr Ala Glu Asn Val 150 155 160 165 gct aaa gag tac gga att acc cgt gaa atg cag gat gaa ctg gcg cta 823Ala Lys Glu Tyr Gly Ile Thr Arg Glu Met Gln Asp Glu Leu Ala Leu 170 175 180 cat tca cag cgt aaa gcg gca gcc gca att gag tcc ggt gct ttt aca 871His Ser Gln Arg Lys Ala Ala Ala Ala Ile Glu Ser Gly Ala Phe Thr 185 190 195 gcc gaa atc gtc ccg gta aat gtt gtc act cga aag aaa acc ttc gtc 919Ala Glu Ile Val Pro Val Asn Val Val Thr Arg Lys Lys Thr Phe Val 200 205 210 ttc agt caa gac gaa ttc ccg aaa gcg aat tca acg gct gaa gcg tta 967Phe Ser Gln Asp Glu Phe Pro Lys Ala Asn Ser Thr Ala Glu Ala Leu 215 220 225 ggt gca ttg cgc ccg gcc ttc gat aaa gca gga aca gtc acc gct ggg 1015Gly Ala Leu Arg Pro Ala Phe Asp Lys Ala Gly Thr Val Thr Ala Gly 230 235 240 245 aac gcg tct ggt att aac gac ggt gct gcc gct ctg gtg att atg gaa 1063Asn Ala Ser Gly Ile Asn Asp Gly Ala Ala Ala Leu Val Ile Met Glu 250 255 260 gaa tct gcg gcg ctg gca gca ggc ctt acc ccc ctg gct cgc att aaa 1111Glu Ser Ala Ala Leu Ala Ala Gly Leu Thr Pro Leu Ala Arg Ile Lys 265 270 275 agt tat gcc agc ggt ggc gtg ccc ccc gca ttg atg ggt atg ggg cca 1159Ser Tyr Ala Ser Gly Gly Val Pro Pro Ala Leu Met Gly Met Gly Pro 280 285 290 gta cct gcc acg caa aaa gcg tta caa ctg gcg ggg ctg caa ctg gcg 1207Val Pro Ala Thr Gln Lys Ala Leu Gln Leu Ala Gly Leu Gln Leu Ala

295 300 305 gat att gat ctc att gag gct aat gaa gca ttt gct gca cag ttc ctt 1255Asp Ile Asp Leu Ile Glu Ala Asn Glu Ala Phe Ala Ala Gln Phe Leu 310 315 320 325 gcc gtt ggg aaa aac ctg ggc ttt gat tct gag aaa gtg aat gtc aac 1303Ala Val Gly Lys Asn Leu Gly Phe Asp Ser Glu Lys Val Asn Val Asn 330 335 340 ggc ggg gcc atc gcg ctc ggg cat cct atc ggt gcc agt ggt gct cgt 1351Gly Gly Ala Ile Ala Leu Gly His Pro Ile Gly Ala Ser Gly Ala Arg 345 350 355 att ctg gtc aca cta tta cat gcc atg cag gca cgc gat aaa acg ctg 1399Ile Leu Val Thr Leu Leu His Ala Met Gln Ala Arg Asp Lys Thr Leu 360 365 370 ggg ctg gca aca ctg tgc att ggc ggc ggt cag gga att gcg atg gtg 1447Gly Leu Ala Thr Leu Cys Ile Gly Gly Gly Gln Gly Ile Ala Met Val 375 380 385 att gaa cgg ttg aat taa ggaggacagc taa atg aaa ctc tca act aaa 1496Ile Glu Arg Leu Asn Met Lys Leu Ser Thr Lys 390 395 400 ctt tgt tgg tgt ggt att aaa gga aga ctt agg ccg caa aag caa caa 1544Leu Cys Trp Cys Gly Ile Lys Gly Arg Leu Arg Pro Gln Lys Gln Gln 405 410 415 caa tta cac aat aca aac ttg caa atg act gaa cta aaa aaa caa aag 1592Gln Leu His Asn Thr Asn Leu Gln Met Thr Glu Leu Lys Lys Gln Lys 420 425 430 acc gct gaa caa aaa acc aga cct caa aat gtc ggt att aaa ggt atc 1640Thr Ala Glu Gln Lys Thr Arg Pro Gln Asn Val Gly Ile Lys Gly Ile 435 440 445 caa att tac atc cca act caa tgt gtc aac caa tct gag cta gag aaa 1688Gln Ile Tyr Ile Pro Thr Gln Cys Val Asn Gln Ser Glu Leu Glu Lys 450 455 460 ttt gat ggc gtt tct caa ggt aaa tac aca att ggt ctg ggc caa acc 1736Phe Asp Gly Val Ser Gln Gly Lys Tyr Thr Ile Gly Leu Gly Gln Thr 465 470 475 480 aac atg tct ttt gtc aat gac aga gaa gat atc tac tcg atg tcc cta 1784Asn Met Ser Phe Val Asn Asp Arg Glu Asp Ile Tyr Ser Met Ser Leu 485 490 495 act gtt ttg tct aag ttg atc aag agt tac aac atc gac acc aac aaa 1832Thr Val Leu Ser Lys Leu Ile Lys Ser Tyr Asn Ile Asp Thr Asn Lys 500 505 510 att ggt aga tta gaa gtc ggt act gaa act ctg att gac aag tcc aag 1880Ile Gly Arg Leu Glu Val Gly Thr Glu Thr Leu Ile Asp Lys Ser Lys 515 520 525 tct gtc aag tct gtc ttg atg caa ttg ttt ggt gaa aac act gac gtc 1928Ser Val Lys Ser Val Leu Met Gln Leu Phe Gly Glu Asn Thr Asp Val 530 535 540 gaa ggt att gac acg ctt aat gcc gct tac ggt ggt acc aac gcg ttg 1976Glu Gly Ile Asp Thr Leu Asn Ala Ala Tyr Gly Gly Thr Asn Ala Leu 545 550 555 560 ttc aac tct ttg aac tgg att gaa tct aac gca tgg gat ggt aga gac 2024Phe Asn Ser Leu Asn Trp Ile Glu Ser Asn Ala Trp Asp Gly Arg Asp 565 570 575 gcc att gta gtt tgc ggt gat att gcc atc tac gat aag ggt gcc gca 2072Ala Ile Val Val Cys Gly Asp Ile Ala Ile Tyr Asp Lys Gly Ala Ala 580 585 590 aga cca acc ggt ggt gcc ggt act gtt gct atg tgg atc ggt cct gat 2120Arg Pro Thr Gly Gly Ala Gly Thr Val Ala Met Trp Ile Gly Pro Asp 595 600 605 gct cca att gta ttt gac tct gta aga gct tct tac atg gaa cac gcc 2168Ala Pro Ile Val Phe Asp Ser Val Arg Ala Ser Tyr Met Glu His Ala 610 615 620 tac gat ttt tac aag cca gat ttc acc agc gaa tat cct tac gtc gat 2216Tyr Asp Phe Tyr Lys Pro Asp Phe Thr Ser Glu Tyr Pro Tyr Val Asp 625 630 635 640 ggt cat ttt tca tta act tgt tac gtc aag gct ctt gat caa gtt tac 2264Gly His Phe Ser Leu Thr Cys Tyr Val Lys Ala Leu Asp Gln Val Tyr 645 650 655 aag agt tat tcc aag aag gct att tct aaa ggg ttg gtt agc gat ccc 2312Lys Ser Tyr Ser Lys Lys Ala Ile Ser Lys Gly Leu Val Ser Asp Pro 660 665 670 gct ggt tcg gat gct ttg aac gtt ttg aaa tat ttc gac tac aac gtt 2360Ala Gly Ser Asp Ala Leu Asn Val Leu Lys Tyr Phe Asp Tyr Asn Val 675 680 685 ttc cat gtt cca acc tgt aaa ttg gtc aca aaa tca tac ggt aga tta 2408Phe His Val Pro Thr Cys Lys Leu Val Thr Lys Ser Tyr Gly Arg Leu 690 695 700 cta tat aac gat ttc aga gcc aat cct caa ttg ttc cca gaa gtt gac 2456Leu Tyr Asn Asp Phe Arg Ala Asn Pro Gln Leu Phe Pro Glu Val Asp 705 710 715 720 gcc gaa tta gct act cgc gat tat gac gaa tct tta acc gat aag aac 2504Ala Glu Leu Ala Thr Arg Asp Tyr Asp Glu Ser Leu Thr Asp Lys Asn 725 730 735 att gaa aaa act ttt gtt aat gtt gct aag cca ttc cac aaa gag aga 2552Ile Glu Lys Thr Phe Val Asn Val Ala Lys Pro Phe His Lys Glu Arg 740 745 750 gtt gcc caa tct ttg att gtt cca aca aac aca ggt aac atg tac acc 2600Val Ala Gln Ser Leu Ile Val Pro Thr Asn Thr Gly Asn Met Tyr Thr 755 760 765 gca tct gtt tat gcc gcc ttt gca tct cta tta aac tat gtt gga tct 2648Ala Ser Val Tyr Ala Ala Phe Ala Ser Leu Leu Asn Tyr Val Gly Ser 770 775 780 gac gac tta caa ggc aag cgt gtt ggt tta ttt tct tac ggt tcc ggt 2696Asp Asp Leu Gln Gly Lys Arg Val Gly Leu Phe Ser Tyr Gly Ser Gly 785 790 795 800 tta gct gca tct cta tat tct tgc aaa att gtt ggt gac gtc caa cat 2744Leu Ala Ala Ser Leu Tyr Ser Cys Lys Ile Val Gly Asp Val Gln His 805 810 815 att atc aag gaa tta gat att act aac aaa tta gcc aag aga atc acc 2792Ile Ile Lys Glu Leu Asp Ile Thr Asn Lys Leu Ala Lys Arg Ile Thr 820 825 830 gaa act cca aag gat tac gaa gct gcc atc gaa ttg aga gaa aat gcc 2840Glu Thr Pro Lys Asp Tyr Glu Ala Ala Ile Glu Leu Arg Glu Asn Ala 835 840 845 cat ttg aag aag aac ttc aaa cct caa ggt tcc att gag cat ttg caa 2888His Leu Lys Lys Asn Phe Lys Pro Gln Gly Ser Ile Glu His Leu Gln 850 855 860 agt ggt gtt tac tac ttg acc aac atc gat gac aaa ttt aga aga tct 2936Ser Gly Val Tyr Tyr Leu Thr Asn Ile Asp Asp Lys Phe Arg Arg Ser 865 870 875 880 tac gat gtt aaa aaa taa ggaggattac act atg gtt tta acc aat aaa 2985Tyr Asp Val Lys Lys Met Val Leu Thr Asn Lys 885 890 aca gtc att tct gga tcg aaa gtc aaa agt tta tca tct gcg caa tcg 3033Thr Val Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser 895 900 905 agc tca tca gga cct tca tca tct agt gag gaa gat gat tcc cgc gat 3081Ser Ser Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp 910 915 920 att gaa agc ttg gat aag aaa ata cgt cct tta gaa gaa tta gaa gca 3129Ile Glu Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala 925 930 935 tta tta agt agt gga aat aca aaa caa ttg aag aac aaa gag gtc gct 3177Leu Leu Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala 940 945 950 955 gcc ttg gtt att cac ggt aag tta cct ttg tac gct ttg gag aaa aaa 3225Ala Leu Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys 960 965 970 tta ggt gat act acg aga gcg gtt gcg gta cgt agg aag gct ctt tca 3273Leu Gly Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser 975 980 985 att ttg gca gaa gct cct gta tta gca tct gat cgt tta cca tat aaa 3321Ile Leu Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys 990 995 1000 aat tat gac tac gac cgc gta ttt ggc gct tgt tgt gaa aat gtt 3366Asn Tyr Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val 1005 1010 1015 ata ggt tac atg cct ttg ccc gtt ggt gtt ata ggc ccc ttg gtt 3411Ile Gly Tyr Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val 1020 1025 1030 atc gat ggt aca tct tat cat ata cca atg gca act aca gag ggt 3456Ile Asp Gly Thr Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly 1035 1040 1045 tgt ttg gta gct tct gcc atg cgt ggc tgt aag gca atc aat gct 3501Cys Leu Val Ala Ser Ala Met Arg Gly Cys Lys Ala Ile Asn Ala 1050 1055 1060 ggc ggt ggt gca aca act gtt tta act aag gat ggt atg aca aga 3546Gly Gly Gly Ala Thr Thr Val Leu Thr Lys Asp Gly Met Thr Arg 1065 1070 1075 ggc cca gta gtc cgt ttc cca act ttg aaa aga tct ggt gcc tgt 3591Gly Pro Val Val Arg Phe Pro Thr Leu Lys Arg Ser Gly Ala Cys 1080 1085 1090 aag ata tgg tta gac tca gaa gag gga caa aac gca att aaa aaa 3636Lys Ile Trp Leu Asp Ser Glu Glu Gly Gln Asn Ala Ile Lys Lys 1095 1100 1105 gct ttt aac tct aca tca aga ttt gca cgt ctg caa cat att caa 3681Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gln His Ile Gln 1110 1115 1120 act tgt cta gca gga gat tta ctc ttc atg aga ttt aga aca act 3726Thr Cys Leu Ala Gly Asp Leu Leu Phe Met Arg Phe Arg Thr Thr 1125 1130 1135 act ggt gac gca atg ggt atg aat atg att tct aaa ggt gtc gaa 3771Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser Lys Gly Val Glu 1140 1145 1150 tac tca tta aag caa atg gta gaa gag tat ggc tgg gaa gat atg 3816Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp Glu Asp Met 1155 1160 1165 gag gtt gtc tcc gtt tct ggt aac tac tgt acc gac aaa aaa cca 3861Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys Lys Pro 1170 1175 1180 gct gcc atc aac tgg atc gaa ggt cgt ggt aag agt gtc gtc gca 3906Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val Ala 1185 1190 1195 gaa gct act att cct ggt gat gtt gtc aga aaa gtg tta aaa agt 3951Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 1200 1205 1210 gat gtt tcc gca ttg gtt gag ttg aac att gct aag aat ttg gtt 3996Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val 1215 1220 1225 gga tct gca atg gct ggg tct gtt ggt gga ttt aac gca cat gca 4041Gly Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala 1230 1235 1240 gct aat tta gtg aca gct gtt ttc ttg gca tta gga caa gat cct 4086Ala Asn Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro 1245 1250 1255 gca caa aat gtt gaa agt tcc aac tgt ata aca ttg atg aaa gaa 4131Ala Gln Asn Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu 1260 1265 1270 gtg gac ggt gat ttg aga att tcc gta tcc atg cca tcc atc gaa 4176Val Asp Gly Asp Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu 1275 1280 1285 gta ggt acc atc ggt ggt ggt act gtt cta gaa cca caa ggt gcc 4221Val Gly Thr Ile Gly Gly Gly Thr Val Leu Glu Pro Gln Gly Ala 1290 1295 1300 atg ttg gac tta tta ggt gta aga ggc ccg cat gct acc gct cct 4266Met Leu Asp Leu Leu Gly Val Arg Gly Pro His Ala Thr Ala Pro 1305 1310 1315 ggt acc aac gca cgt caa tta gca aga ata gtt gcc tgt gcc gtc 4311Gly Thr Asn Ala Arg Gln Leu Ala Arg Ile Val Ala Cys Ala Val 1320 1325 1330 ttg gca ggt gaa tta tcc tta tgt gct gcc cta gca gcc ggc cat 4356Leu Ala Gly Glu Leu Ser Leu Cys Ala Ala Leu Ala Ala Gly His 1335 1340 1345 ttg gtt caa agt cat atg acc cac aac agg aaa cct gct gaa cca 4401Leu Val Gln Ser His Met Thr His Asn Arg Lys Pro Ala Glu Pro 1350 1355 1360 aca aaa cct aac aat ttg gac gcc act gat ata aat cgt ttg aaa 4446Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp Ile Asn Arg Leu Lys 1365 1370 1375 gat ggg tcc gtc acc tgc att aaa tcc taagtcgacc tgcaggcatg 4493Asp Gly Ser Val Thr Cys Ile Lys Ser 1380 1385 caagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga 4553acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc 4613tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc 4673ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact 4733gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc 4793cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc 4853cataaactgc caggcatcaa attaagcaga aggccatcct gacggtaccg ctaccagcgg 4913tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 4973gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 5033actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gggcatttga 5093gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata 5153agcggctatt taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt 5213ctgccattca tccgcttatt atcacttatt caggcgtagc accaggcgtt taagggcacc 5273aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt 5333cattaagcat tctgccgaca tggaagccat cacagacggc atgatgaacc tgaatcgcca 5393gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga 5453agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg 5513ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt 5573aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac 5633tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac 5693tatcccatat caccagctca ccgtctttca ttgccatacg gaattccgga tgagcattca 5753tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg 5813tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg 5873actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc 5933cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa 5993atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat 6053caacgtctca ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag 6113gatttattta ttctgcgaag tgatcttccg tcacaggtat ttattcggcg caaagtgcgt 6173cgggtgatgc tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg 6233gcttctgttt ctatcagctg tccctcctgt tcagctactg acggggtggt gcgtaacggc 6293aaaagcaccg ccggacatca gcgctagcgg agtgtatact ggcttactat gttggcactg 6353atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca 6413gcagaatatg tgatacagga tatattccgc ttcctcgctc actgactcgc tacgctcggt 6473cgttcgactg cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc 6533caggaagata cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc 6593cgcccccctg acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca 6653ggactataaa gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct 6713gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct 6773gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt 6833tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca

6893tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt 6953catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc 7013agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg 7073cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc 7133atcttattaa tcagataaaa tatttgctca tgagcccgaa gtggcgagcc cgatcttccc 7193catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg 7253ccacgatgcg tccggcgtag aggatctgct catgtttgac agcttatc 730122394PRTEscherichia coli 22Met Lys Asn Cys Val Ile Val Ser Ala Val Arg Thr Ala Ile Gly Ser 1 5 10 15 Phe Asn Gly Ser Leu Ala Ser Thr Ser Ala Ile Asp Leu Gly Ala Thr 20 25 30 Val Ile Lys Ala Ala Ile Glu Arg Ala Lys Ile Asp Ser Gln His Val 35 40 45 Asp Glu Val Ile Met Gly Asn Val Leu Gln Ala Gly Leu Gly Gln Asn 50 55 60 Pro Ala Arg Gln Ala Leu Leu Lys Ser Gly Leu Ala Glu Thr Val Cys 65 70 75 80 Gly Phe Thr Val Asn Lys Val Cys Gly Ser Gly Leu Lys Ser Val Ala 85 90 95 Leu Ala Ala Gln Ala Ile Gln Ala Gly Gln Ala Gln Ser Ile Val Ala 100 105 110 Gly Gly Met Glu Asn Met Ser Leu Ala Pro Tyr Leu Leu Asp Ala Lys 115 120 125 Ala Arg Ser Gly Tyr Arg Leu Gly Asp Gly Gln Val Tyr Asp Val Ile 130 135 140 Leu Arg Asp Gly Leu Met Cys Ala Thr His Gly Tyr His Met Gly Ile 145 150 155 160 Thr Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu Met Gln 165 170 175 Asp Glu Leu Ala Leu His Ser Gln Arg Lys Ala Ala Ala Ala Ile Glu 180 185 190 Ser Gly Ala Phe Thr Ala Glu Ile Val Pro Val Asn Val Val Thr Arg 195 200 205 Lys Lys Thr Phe Val Phe Ser Gln Asp Glu Phe Pro Lys Ala Asn Ser 210 215 220 Thr Ala Glu Ala Leu Gly Ala Leu Arg Pro Ala Phe Asp Lys Ala Gly 225 230 235 240 Thr Val Thr Ala Gly Asn Ala Ser Gly Ile Asn Asp Gly Ala Ala Ala 245 250 255 Leu Val Ile Met Glu Glu Ser Ala Ala Leu Ala Ala Gly Leu Thr Pro 260 265 270 Leu Ala Arg Ile Lys Ser Tyr Ala Ser Gly Gly Val Pro Pro Ala Leu 275 280 285 Met Gly Met Gly Pro Val Pro Ala Thr Gln Lys Ala Leu Gln Leu Ala 290 295 300 Gly Leu Gln Leu Ala Asp Ile Asp Leu Ile Glu Ala Asn Glu Ala Phe 305 310 315 320 Ala Ala Gln Phe Leu Ala Val Gly Lys Asn Leu Gly Phe Asp Ser Glu 325 330 335 Lys Val Asn Val Asn Gly Gly Ala Ile Ala Leu Gly His Pro Ile Gly 340 345 350 Ala Ser Gly Ala Arg Ile Leu Val Thr Leu Leu His Ala Met Gln Ala 355 360 365 Arg Asp Lys Thr Leu Gly Leu Ala Thr Leu Cys Ile Gly Gly Gly Gln 370 375 380 Gly Ile Ala Met Val Ile Glu Arg Leu Asn 385 390 23491PRTEscherichia coli 23Met Lys Leu Ser Thr Lys Leu Cys Trp Cys Gly Ile Lys Gly Arg Leu 1 5 10 15 Arg Pro Gln Lys Gln Gln Gln Leu His Asn Thr Asn Leu Gln Met Thr 20 25 30 Glu Leu Lys Lys Gln Lys Thr Ala Glu Gln Lys Thr Arg Pro Gln Asn 35 40 45 Val Gly Ile Lys Gly Ile Gln Ile Tyr Ile Pro Thr Gln Cys Val Asn 50 55 60 Gln Ser Glu Leu Glu Lys Phe Asp Gly Val Ser Gln Gly Lys Tyr Thr 65 70 75 80 Ile Gly Leu Gly Gln Thr Asn Met Ser Phe Val Asn Asp Arg Glu Asp 85 90 95 Ile Tyr Ser Met Ser Leu Thr Val Leu Ser Lys Leu Ile Lys Ser Tyr 100 105 110 Asn Ile Asp Thr Asn Lys Ile Gly Arg Leu Glu Val Gly Thr Glu Thr 115 120 125 Leu Ile Asp Lys Ser Lys Ser Val Lys Ser Val Leu Met Gln Leu Phe 130 135 140 Gly Glu Asn Thr Asp Val Glu Gly Ile Asp Thr Leu Asn Ala Ala Tyr 145 150 155 160 Gly Gly Thr Asn Ala Leu Phe Asn Ser Leu Asn Trp Ile Glu Ser Asn 165 170 175 Ala Trp Asp Gly Arg Asp Ala Ile Val Val Cys Gly Asp Ile Ala Ile 180 185 190 Tyr Asp Lys Gly Ala Ala Arg Pro Thr Gly Gly Ala Gly Thr Val Ala 195 200 205 Met Trp Ile Gly Pro Asp Ala Pro Ile Val Phe Asp Ser Val Arg Ala 210 215 220 Ser Tyr Met Glu His Ala Tyr Asp Phe Tyr Lys Pro Asp Phe Thr Ser 225 230 235 240 Glu Tyr Pro Tyr Val Asp Gly His Phe Ser Leu Thr Cys Tyr Val Lys 245 250 255 Ala Leu Asp Gln Val Tyr Lys Ser Tyr Ser Lys Lys Ala Ile Ser Lys 260 265 270 Gly Leu Val Ser Asp Pro Ala Gly Ser Asp Ala Leu Asn Val Leu Lys 275 280 285 Tyr Phe Asp Tyr Asn Val Phe His Val Pro Thr Cys Lys Leu Val Thr 290 295 300 Lys Ser Tyr Gly Arg Leu Leu Tyr Asn Asp Phe Arg Ala Asn Pro Gln 305 310 315 320 Leu Phe Pro Glu Val Asp Ala Glu Leu Ala Thr Arg Asp Tyr Asp Glu 325 330 335 Ser Leu Thr Asp Lys Asn Ile Glu Lys Thr Phe Val Asn Val Ala Lys 340 345 350 Pro Phe His Lys Glu Arg Val Ala Gln Ser Leu Ile Val Pro Thr Asn 355 360 365 Thr Gly Asn Met Tyr Thr Ala Ser Val Tyr Ala Ala Phe Ala Ser Leu 370 375 380 Leu Asn Tyr Val Gly Ser Asp Asp Leu Gln Gly Lys Arg Val Gly Leu 385 390 395 400 Phe Ser Tyr Gly Ser Gly Leu Ala Ala Ser Leu Tyr Ser Cys Lys Ile 405 410 415 Val Gly Asp Val Gln His Ile Ile Lys Glu Leu Asp Ile Thr Asn Lys 420 425 430 Leu Ala Lys Arg Ile Thr Glu Thr Pro Lys Asp Tyr Glu Ala Ala Ile 435 440 445 Glu Leu Arg Glu Asn Ala His Leu Lys Lys Asn Phe Lys Pro Gln Gly 450 455 460 Ser Ile Glu His Leu Gln Ser Gly Val Tyr Tyr Leu Thr Asn Ile Asp 465 470 475 480 Asp Lys Phe Arg Arg Ser Tyr Asp Val Lys Lys 485 490 24502PRTEscherichia coli 24Met Val Leu Thr Asn Lys Thr Val Ile Ser Gly Ser Lys Val Lys Ser 1 5 10 15 Leu Ser Ser Ala Gln Ser Ser Ser Ser Gly Pro Ser Ser Ser Ser Glu 20 25 30 Glu Asp Asp Ser Arg Asp Ile Glu Ser Leu Asp Lys Lys Ile Arg Pro 35 40 45 Leu Glu Glu Leu Glu Ala Leu Leu Ser Ser Gly Asn Thr Lys Gln Leu 50 55 60 Lys Asn Lys Glu Val Ala Ala Leu Val Ile His Gly Lys Leu Pro Leu 65 70 75 80 Tyr Ala Leu Glu Lys Lys Leu Gly Asp Thr Thr Arg Ala Val Ala Val 85 90 95 Arg Arg Lys Ala Leu Ser Ile Leu Ala Glu Ala Pro Val Leu Ala Ser 100 105 110 Asp Arg Leu Pro Tyr Lys Asn Tyr Asp Tyr Asp Arg Val Phe Gly Ala 115 120 125 Cys Cys Glu Asn Val Ile Gly Tyr Met Pro Leu Pro Val Gly Val Ile 130 135 140 Gly Pro Leu Val Ile Asp Gly Thr Ser Tyr His Ile Pro Met Ala Thr 145 150 155 160 Thr Glu Gly Cys Leu Val Ala Ser Ala Met Arg Gly Cys Lys Ala Ile 165 170 175 Asn Ala Gly Gly Gly Ala Thr Thr Val Leu Thr Lys Asp Gly Met Thr 180 185 190 Arg Gly Pro Val Val Arg Phe Pro Thr Leu Lys Arg Ser Gly Ala Cys 195 200 205 Lys Ile Trp Leu Asp Ser Glu Glu Gly Gln Asn Ala Ile Lys Lys Ala 210 215 220 Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gln His Ile Gln Thr Cys 225 230 235 240 Leu Ala Gly Asp Leu Leu Phe Met Arg Phe Arg Thr Thr Thr Gly Asp 245 250 255 Ala Met Gly Met Asn Met Ile Ser Lys Gly Val Glu Tyr Ser Leu Lys 260 265 270 Gln Met Val Glu Glu Tyr Gly Trp Glu Asp Met Glu Val Val Ser Val 275 280 285 Ser Gly Asn Tyr Cys Thr Asp Lys Lys Pro Ala Ala Ile Asn Trp Ile 290 295 300 Glu Gly Arg Gly Lys Ser Val Val Ala Glu Ala Thr Ile Pro Gly Asp 305 310 315 320 Val Val Arg Lys Val Leu Lys Ser Asp Val Ser Ala Leu Val Glu Leu 325 330 335 Asn Ile Ala Lys Asn Leu Val Gly Ser Ala Met Ala Gly Ser Val Gly 340 345 350 Gly Phe Asn Ala His Ala Ala Asn Leu Val Thr Ala Val Phe Leu Ala 355 360 365 Leu Gly Gln Asp Pro Ala Gln Asn Val Glu Ser Ser Asn Cys Ile Thr 370 375 380 Leu Met Lys Glu Val Asp Gly Asp Leu Arg Ile Ser Val Ser Met Pro 385 390 395 400 Ser Ile Glu Val Gly Thr Ile Gly Gly Gly Thr Val Leu Glu Pro Gln 405 410 415 Gly Ala Met Leu Asp Leu Leu Gly Val Arg Gly Pro His Ala Thr Ala 420 425 430 Pro Gly Thr Asn Ala Arg Gln Leu Ala Arg Ile Val Ala Cys Ala Val 435 440 445 Leu Ala Gly Glu Leu Ser Leu Cys Ala Ala Leu Ala Ala Gly His Leu 450 455 460 Val Gln Ser His Met Thr His Asn Arg Lys Pro Ala Glu Pro Thr Lys 465 470 475 480 Pro Asn Asn Leu Asp Ala Thr Asp Ile Asn Arg Leu Lys Asp Gly Ser 485 490 495 Val Thr Cys Ile Lys Ser 500 252283DNAEscherichia colimisc_feature(329)..(363)FRT sitemisc_feature(1551)..(1585)FRT sitepromoter(1907)..(1979)pBAD promoterCDS(1991)..(2281)folp gene excerpt 25aagatgctct catgaaatat gagactatcg acgcaccgca gattgatgac ctgatggcac 60gtcgcgatgt acgtccgcca gcgggctggg aagaaccagg cgcttctaac aattctggcg 120acaatggtag tccaaaggct cctcgtccgg ttgatgaacc gcgtacgccg aacccgggta 180acaccatgtc agagcagtta ggcgacaagt aagttcccgc atcagatgac tgtatttgta 240ccgaaaaccc cggggcgtgc tccggggttt tttcttatca attcatacca gggataacat 300cagcgattgt gtaggctgga gctgcttcga agttcctata ctttctagag aataggaact 360tcggaatagg aacttcaaga tccccttatt agaagaactc gtcaagaagg cgatagaagg 420cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt 480cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg 540ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat 600tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct 660tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct 720gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt 780ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga 840tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc 900ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa 960cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac 1020cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg 1080cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc 1140aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc 1200ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca 1260tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg 1320gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag 1380ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat 1440tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc 1500agcccttgcg ccctgagtgc ttgcggcagc gtgagcttca aaagcgctct gaagttccta 1560tactttctag agaataggaa cttcgaactg caggtcgacg gatccccgga atattcatat 1620gtgcgcttca gccatacttt tcatactccc gccattcaga gaagaaacca attgtccata 1680ttgcatcaga cattgccgtc actgcgtctt ttactggctc ttctcgctaa ccaaaccggt 1740aaccccgctt attaaaagca ttctgtaaca aagcgggacc aaagccatga caaaaacgcg 1800taacaaaagt gtctataatc acggcagaaa agtccacatt gattatttgc acggcgtcac 1860actttgctat gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt 1920atcgcaactc tctactgttt ctccataccc gtttttttgg gctagcgaat tcgagctcga 1980cttggaaggt atg aaa ctc ttt gcc cag ggt act tca ctg gac ctt agc 2029 Met Lys Leu Phe Ala Gln Gly Thr Ser Leu Asp Leu Ser 1 5 10 cat cct cac gta atg ggg atc ctc aac gtc acg cct gat tcc ttt tcg 2077His Pro His Val Met Gly Ile Leu Asn Val Thr Pro Asp Ser Phe Ser 15 20 25 gat ggt ggc acg cat aac tcg ctg ata gat gcg gtg aaa cat gcg aat 2125Asp Gly Gly Thr His Asn Ser Leu Ile Asp Ala Val Lys His Ala Asn 30 35 40 45 ctg atg atc aac gct ggc gcg acg atc att gac gtt ggt ggc gag tcc 2173Leu Met Ile Asn Ala Gly Ala Thr Ile Ile Asp Val Gly Gly Glu Ser 50 55 60 acg cgc cca ggg gcg gcg gaa gtt agc gtt gaa gaa gag ttg caa cgt 2221Thr Arg Pro Gly Ala Ala Glu Val Ser Val Glu Glu Glu Leu Gln Arg 65 70 75 gtt att cct gtg gtt gag gca att gct caa cgc ttc gaa gtc tgg atc 2269Val Ile Pro Val Val Glu Ala Ile Ala Gln Arg Phe Glu Val Trp Ile 80 85 90 tca gtc gat aca tc 2283Ser Val Asp Thr 95 2697PRTEscherichia coli 26Met Lys Leu Phe Ala Gln Gly Thr Ser Leu Asp Leu Ser His Pro His 1 5 10 15 Val Met Gly Ile Leu Asn Val Thr Pro Asp Ser Phe Ser Asp Gly Gly 20 25 30 Thr His Asn Ser Leu Ile Asp Ala Val Lys His Ala Asn Leu Met Ile 35 40 45 Asn Ala Gly Ala Thr Ile Ile Asp Val Gly Gly Glu Ser Thr Arg Pro 50 55 60 Gly Ala Ala Glu Val Ser Val Glu Glu Glu Leu Gln Arg Val Ile Pro 65 70 75 80 Val Val Glu Ala Ile Ala Gln Arg Phe Glu Val Trp Ile Ser Val Asp 85 90 95 Thr 27795DNAEscherichia coliCDS(1)..(795)kanR gene encoding Neomycin phosphotransferase II 27atg att gaa caa gat gga ttg cac gca ggt tct ccg gcc gct tgg gtg 48Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val 1 5 10 15 gag agg cta ttc ggc tat gac tgg gca caa cag aca atc ggc tgc tct 96Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser 20 25 30 gat gcc gcc gtg ttc cgg ctg tca gcg cag ggg cgc ccg gtt ctt ttt 144Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe 35 40 45 gtc aag acc gac ctg tcc ggt gcc ctg aat gaa ctg cag gac gag gca 192Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala 50 55 60 gcg cgg cta tcg tgg ctg gcc acg acg ggc gtt cct tgc gca gct gtg 240Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val 65 70 75 80 ctc gac gtt gtc act gaa gcg gga agg gac tgg ctg cta ttg ggc gaa 288Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu 85 90 95 gtg ccg ggg cag gat ctc ctg tca tct cac ctt gct cct gcc gag aaa 336Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys 100 105 110 gta tcc atc atg gct gat gca atg cgg cgg ctg cat acg ctt gat ccg 384Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro 115 120 125

gct acc tgc cca ttc gac cac caa gcg aaa cat cgc atc gag cga gca 432Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala 130 135 140 cgt act cgg atg gaa gcc ggt ctt gtc gat cag gat gat ctg gac gaa 480Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu 145 150 155 160 gag cat cag ggg ctc gcg cca gcc gaa ctg ttc gcc agg ctc aag gcg 528Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala 165 170 175 cgc atg ccc gac ggc gag gat ctc gtc gtg acc cat ggc gat gcc tgc 576Arg Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys 180 185 190 ttg ccg aat atc atg gtg gaa aat ggc cgc ttt tct gga ttc atc gac 624Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp 195 200 205 tgt ggc cgg ctg ggt gtg gcg gac cgc tat cag gac ata gcg ttg gct 672Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala 210 215 220 acc cgt gat att gct gaa gag ctt ggc ggc gaa tgg gct gac cgc ttc 720Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe 225 230 235 240 ctc gtg ctt tac ggt atc gcc gct ccc gat tcg cag cgc atc gcc ttc 768Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe 245 250 255 tat cgc ctt ctt gac gag ttc ttc taa 795Tyr Arg Leu Leu Asp Glu Phe Phe 260 28264PRTEscherichia coli 28Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val 1 5 10 15 Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser 20 25 30 Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe 35 40 45 Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala 50 55 60 Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val 65 70 75 80 Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu 85 90 95 Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys 100 105 110 Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro 115 120 125 Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala 130 135 140 Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu 145 150 155 160 Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala 165 170 175 Arg Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys 180 185 190 Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp 195 200 205 Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala 210 215 220 Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe 225 230 235 240 Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe 245 250 255 Tyr Arg Leu Leu Asp Glu Phe Phe 260 294739DNAEscherichia coliCDS(1894)..(2865)specR gene encoding aminoglycoside nucleotidyltransferaserep_origin(3459)..(4078)Origin of replication 29atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660tgccagtagg cgcgcggacg aaagtaaacc cactgtcgat accattcgcg agcctccgga 720tgacgaccca agtgcagaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900gggagtaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcgaatt cgagctcgag 1320gaggaaacca tggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggct 1380gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg 1440gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc 1500cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag 1560tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt 1620tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat 1680ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc 1740aggcatcaaa ttaagcagaa ggccatcctg acggtgatat ccggatgaag gcacgaaccc 1800agtggacata agcctcgttc ggttcgtaag ctgtaatgca agtagcgtaa ctgccgtcac 1860gcaactggtc cacaaccttg accgaacgca gcg gtg gta acg gcg cag tgg cgg 1914 Val Val Thr Ala Gln Trp Arg 1 5 ttt tca tgg ctt ctt gtt atg aca tgt ttt ttt ggg gta cag tct atg 1962Phe Ser Trp Leu Leu Val Met Thr Cys Phe Phe Gly Val Gln Ser Met 10 15 20 cct cgg gca tcc aag cag caa gcg cgt tac gcc gtg ggt cga tgt ttg 2010Pro Arg Ala Ser Lys Gln Gln Ala Arg Tyr Ala Val Gly Arg Cys Leu 25 30 35 atg tta tgg agc agc aac gat gtt acg cag cag ggc agt cgc cct aaa 2058Met Leu Trp Ser Ser Asn Asp Val Thr Gln Gln Gly Ser Arg Pro Lys 40 45 50 55 aca aag tta aac atc atg agg gaa gcg gtg atc gcc gaa gta tcg act 2106Thr Lys Leu Asn Ile Met Arg Glu Ala Val Ile Ala Glu Val Ser Thr 60 65 70 caa cta tca gag gta gtt ggc gtc atc gag cgc cat ctc gaa ccg acg 2154Gln Leu Ser Glu Val Val Gly Val Ile Glu Arg His Leu Glu Pro Thr 75 80 85 ttg ctg gcc gta cat ttg tac ggc tcc gca gtg gat ggc ggc ctg aag 2202Leu Leu Ala Val His Leu Tyr Gly Ser Ala Val Asp Gly Gly Leu Lys 90 95 100 cca cac agt gat att gat ttg ctg gtt acg gtg acc gta agg ctt gat 2250Pro His Ser Asp Ile Asp Leu Leu Val Thr Val Thr Val Arg Leu Asp 105 110 115 gaa aca acg cgg cga gct ttg atc aac gac ctt ttg gaa act tcg gct 2298Glu Thr Thr Arg Arg Ala Leu Ile Asn Asp Leu Leu Glu Thr Ser Ala 120 125 130 135 tcc cct gga gag agc gag att ctc cgc gct gta gaa gtc acc att gtt 2346Ser Pro Gly Glu Ser Glu Ile Leu Arg Ala Val Glu Val Thr Ile Val 140 145 150 gtg cac gac gac atc att ccg tgg cgt tat cca gct aag cgc gaa ctg 2394Val His Asp Asp Ile Ile Pro Trp Arg Tyr Pro Ala Lys Arg Glu Leu 155 160 165 caa ttt gga gaa tgg cag cgc aat gac att ctt gca ggt atc ttc gag 2442Gln Phe Gly Glu Trp Gln Arg Asn Asp Ile Leu Ala Gly Ile Phe Glu 170 175 180 cca gcc acg tac gac att gat ctg gct atc ttg ctg aca aaa gca aga 2490Pro Ala Thr Tyr Asp Ile Asp Leu Ala Ile Leu Leu Thr Lys Ala Arg 185 190 195 gaa cat agc gtt gcc ttg gta ggt cca gcg gcg gag gaa ctc ttt gat 2538Glu His Ser Val Ala Leu Val Gly Pro Ala Ala Glu Glu Leu Phe Asp 200 205 210 215 ccg gtt cct gaa cag gat cta ttt gag gcg cta aat gaa acc tta acg 2586Pro Val Pro Glu Gln Asp Leu Phe Glu Ala Leu Asn Glu Thr Leu Thr 220 225 230 cta tgg aac tcg ccg ccc gac tgg gct ggc gat gag cga aat gta gtg 2634Leu Trp Asn Ser Pro Pro Asp Trp Ala Gly Asp Glu Arg Asn Val Val 235 240 245 ctt acg ttg tcc cgc att tgg tac agc gca gta acc ggc aaa atc gcg 2682Leu Thr Leu Ser Arg Ile Trp Tyr Ser Ala Val Thr Gly Lys Ile Ala 250 255 260 ccg aag gat gtc gct gcc gac tgg gca atg gag cgc ctg ccg gcc cag 2730Pro Lys Asp Val Ala Ala Asp Trp Ala Met Glu Arg Leu Pro Ala Gln 265 270 275 tat cag ccc gtc ata ctt gaa gct aga cag gct tat ctt gga caa gaa 2778Tyr Gln Pro Val Ile Leu Glu Ala Arg Gln Ala Tyr Leu Gly Gln Glu 280 285 290 295 gaa gat cgc ttg gcc tcg cgc gca gat cag ttg gaa gaa ttt gtc cac 2826Glu Asp Arg Leu Ala Ser Arg Ala Asp Gln Leu Glu Glu Phe Val His 300 305 310 tac gtg aaa ggc gag atc acc aag gta gtc ggc aaa taa tgtctaacaa 2875Tyr Val Lys Gly Glu Ile Thr Lys Val Val Gly Lys 315 320 ttcgttcaag ccgacggata tctagattga tttacgcgcc ctgtagcggc gcattaagcg 2935cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 2995ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3055taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 3115aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 3175ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact tgaacaacac 3235tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt 3295ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 3355ttacaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 3415taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 3475tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 3535gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 3595agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 3655aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 3715gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 3775gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 3835tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 3895agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 3955cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 4015gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 4075gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 4135ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 4195cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 4255cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt 4315acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 4375gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 4435tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 4495ggttttcacc gtcatcaccg aaacgcgcga ggcagcaagg agatggcgcc caacagtccc 4555ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4615cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4675gcgccggtga tgccggccac gatgcgtccg gcgtagagga tctgctcatg tttgacagct 4735tatc 473930323PRTEscherichia coli 30Val Val Thr Ala Gln Trp Arg Phe Ser Trp Leu Leu Val Met Thr Cys 1 5 10 15 Phe Phe Gly Val Gln Ser Met Pro Arg Ala Ser Lys Gln Gln Ala Arg 20 25 30 Tyr Ala Val Gly Arg Cys Leu Met Leu Trp Ser Ser Asn Asp Val Thr 35 40 45 Gln Gln Gly Ser Arg Pro Lys Thr Lys Leu Asn Ile Met Arg Glu Ala 50 55 60 Val Ile Ala Glu Val Ser Thr Gln Leu Ser Glu Val Val Gly Val Ile 65 70 75 80 Glu Arg His Leu Glu Pro Thr Leu Leu Ala Val His Leu Tyr Gly Ser 85 90 95 Ala Val Asp Gly Gly Leu Lys Pro His Ser Asp Ile Asp Leu Leu Val 100 105 110 Thr Val Thr Val Arg Leu Asp Glu Thr Thr Arg Arg Ala Leu Ile Asn 115 120 125 Asp Leu Leu Glu Thr Ser Ala Ser Pro Gly Glu Ser Glu Ile Leu Arg 130 135 140 Ala Val Glu Val Thr Ile Val Val His Asp Asp Ile Ile Pro Trp Arg 145 150 155 160 Tyr Pro Ala Lys Arg Glu Leu Gln Phe Gly Glu Trp Gln Arg Asn Asp 165 170 175 Ile Leu Ala Gly Ile Phe Glu Pro Ala Thr Tyr Asp Ile Asp Leu Ala 180 185 190 Ile Leu Leu Thr Lys Ala Arg Glu His Ser Val Ala Leu Val Gly Pro 195 200 205 Ala Ala Glu Glu Leu Phe Asp Pro Val Pro Glu Gln Asp Leu Phe Glu 210 215 220 Ala Leu Asn Glu Thr Leu Thr Leu Trp Asn Ser Pro Pro Asp Trp Ala 225 230 235 240 Gly Asp Glu Arg Asn Val Val Leu Thr Leu Ser Arg Ile Trp Tyr Ser 245 250 255 Ala Val Thr Gly Lys Ile Ala Pro Lys Asp Val Ala Ala Asp Trp Ala 260 265 270 Met Glu Arg Leu Pro Ala Gln Tyr Gln Pro Val Ile Leu Glu Ala Arg 275 280 285 Gln Ala Tyr Leu Gly Gln Glu Glu Asp Arg Leu Ala Ser Arg Ala Asp 290 295 300 Gln Leu Glu Glu Phe Val His Tyr Val Lys Gly Glu Ile Thr Lys Val 305 310 315 320 Val Gly Lys 315507DNAEscherichia colimisc_feature(1291)..(1452)ThiMN15#19 riboswitchCDS(1408)..(1704)tetA-yefM gene encoding tetA leader [nt 1408-1452] fused to yefM antitoxin [nt 1453-1704]CDS(1968)..(2222)yoeB gene encoding yoeB toxinCDS(2775)..(3635)ampR gene encoding beta-lactamaserep_origin(3694)..(3993)f1_origin of replicationrep_origin(4227)..(4846)pBR322_origin of replication 31atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga 720tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260tcgcaactct ctactgtttc tccatacccg aattcaacca aacgactcgg ggtgcccttc 1320tgcgtgaagg ctgagaaata cccgtatcac ctgatctgga taatgccagc gtagggaagc 1380tcttaagaat cagatcagga gcaaact atg caa gtc gac ctg ctg gat cca aaa 1434 Met Gln Val Asp Leu Leu Asp Pro Lys

1 5 tct aac aat gcg ctc atc atg cgt aca att agc tac agc gaa gcg cgt 1482Ser Asn Asn Ala Leu Ile Met Arg Thr Ile Ser Tyr Ser Glu Ala Arg 10 15 20 25 cag aat ttg tcg gca aca atg atg aaa gcc gtt gaa gat cat gcc ccg 1530Gln Asn Leu Ser Ala Thr Met Met Lys Ala Val Glu Asp His Ala Pro 30 35 40 atc ctt att act cgt cag aat gga gag gct tgt gtt ctg atg tca ctc 1578Ile Leu Ile Thr Arg Gln Asn Gly Glu Ala Cys Val Leu Met Ser Leu 45 50 55 gaa gaa tac aac tcg ctg gaa gag acg gct tat cta ctg cgc tcc ccc 1626Glu Glu Tyr Asn Ser Leu Glu Glu Thr Ala Tyr Leu Leu Arg Ser Pro 60 65 70 gct aac gcc cgg aga ttg atg gac tca atc gat agc ctg aaa tca ggc 1674Ala Asn Ala Arg Arg Leu Met Asp Ser Ile Asp Ser Leu Lys Ser Gly 75 80 85 aaa gga acg gaa aag gac atc att gag tga ggcaatcagc tgttgcccgt 1724Lys Gly Thr Glu Lys Asp Ile Ile Glu 90 95 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc 1784gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg 1844agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta 1904tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaagaa 1964acc atg aaa cta atc tgg tct gag gaa tca tgg gac gat tat ctg tac 2012 Met Lys Leu Ile Trp Ser Glu Glu Ser Trp Asp Asp Tyr Leu Tyr 100 105 110 tgg cag gaa aca gat aag cga att gtt aaa aag atc aat gaa ctt atc 2060Trp Gln Glu Thr Asp Lys Arg Ile Val Lys Lys Ile Asn Glu Leu Ile 115 120 125 aaa gat acc cgc aga acg cca ttt gaa ggt aag ggg aag cca gaa ccc 2108Lys Asp Thr Arg Arg Thr Pro Phe Glu Gly Lys Gly Lys Pro Glu Pro 130 135 140 145 ctg aaa cat aat ttg tca ggt ttc tgg tcc cga cgc att aca gag gag 2156Leu Lys His Asn Leu Ser Gly Phe Trp Ser Arg Arg Ile Thr Glu Glu 150 155 160 cac cgt ctg gta tac gcg gtt acc gac gat tca ctg ctc att gca gca 2204His Arg Leu Val Tyr Ala Val Thr Asp Asp Ser Leu Leu Ile Ala Ala 165 170 175 tgt cgt tat cat tat tga atcctctaga gtcgacctgc aggcatgcaa 2252Cys Arg Tyr His Tyr 180 gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg 2312cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga 2372ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca 2432tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg 2492cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg 2552gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat 2612aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc 2672tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 2732taaccctgat aaatgcttca ataatattga aaaaggaaga gt atg agt att caa 2786 Met Ser Ile Gln 185 cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca ttt tgc ctt cct 2834His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro 190 195 200 gtt ttt gct cac cca gaa acg ctg gtg aaa gta aaa gat gct gaa gat 2882Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp 205 210 215 cag ttg ggt gca cga gtg ggt tac atc gaa ctg gat ctc aac agc ggt 2930Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly 220 225 230 aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc 2978Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser 235 240 245 250 act ttt aaa gtt ctg cta tgt ggc gcg gta tta tcc cgt gtt gac gcc 3026Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Val Asp Ala 255 260 265 ggg caa gag caa ctc ggt cgc cgc ata cac tat tct cag aat gac ttg 3074Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu 270 275 280 gtt gag tac tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca 3122Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr 285 290 295 gta aga gaa tta tgc agt gct gcc ata acc atg agt gat aac act gcg 3170Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala 300 305 310 gcc aac tta ctt ctg aca acg atc gga gga ccg aag gag cta acc gct 3218Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala 315 320 325 330 ttt ttg cac aac atg ggg gat cat gta act cgc ctt gat cgt tgg gaa 3266Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu 335 340 345 ccg gag ctg aat gaa gcc ata cca aac gac gag cgt gac acc acg atg 3314Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met 350 355 360 cct gca gca atg gca aca acg ttg cgc aaa cta tta act ggc gaa cta 3362Pro Ala Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu 365 370 375 ctt act cta gct tcc cgg caa caa tta ata gac tgg atg gag gcg gat 3410Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp 380 385 390 aaa gtt gca gga cca ctt ctg cgc tcg gcc ctt ccg gct ggc tgg ttt 3458Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe 395 400 405 410 att gct gat aaa tct gga gcc ggt gag cgt ggg tct cgc ggt atc att 3506Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile 415 420 425 gca gca ctg ggg cca gat ggt aag ccc tcc cgt atc gta gtt atc tac 3554Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr 430 435 440 acg acg ggg agt cag gca act atg gat gaa cga aat aga cag atc gct 3602Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala 445 450 455 gag ata ggt gcc tca ctg att aag cat tgg taa ctgtcagacc aagtttactc 3655Glu Ile Gly Ala Ser Leu Ile Lys His Trp 460 465 atatatactt tagattgatt tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 3715tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 3775tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 3835tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg 3895gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 3955agtccacgtt ctttaatagt ggactcttgt tccaaacttg aacaacactc aaccctatct 4015cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 4075agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttaaa 4135aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 4195tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 4255tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 4315ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 4375ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 4435gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 4495aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 4555ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 4615agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 4675aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 4735aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 4795ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 4855cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 4915tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 4975accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc 5035cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 5095gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg 5155cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 5215ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 5275catcaccgaa acgcgcgagg cagcaaggag atggcgccca acagtccccc ggccacgggg 5335cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct 5395tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg 5455ccggccacga tgcgtccggc gtagaggatc tgctcatgtt tgacagctta tc 55073298PRTEscherichia coli 32Met Gln Val Asp Leu Leu Asp Pro Lys Ser Asn Asn Ala Leu Ile Met 1 5 10 15 Arg Thr Ile Ser Tyr Ser Glu Ala Arg Gln Asn Leu Ser Ala Thr Met 20 25 30 Met Lys Ala Val Glu Asp His Ala Pro Ile Leu Ile Thr Arg Gln Asn 35 40 45 Gly Glu Ala Cys Val Leu Met Ser Leu Glu Glu Tyr Asn Ser Leu Glu 50 55 60 Glu Thr Ala Tyr Leu Leu Arg Ser Pro Ala Asn Ala Arg Arg Leu Met 65 70 75 80 Asp Ser Ile Asp Ser Leu Lys Ser Gly Lys Gly Thr Glu Lys Asp Ile 85 90 95 Ile Glu 3384PRTEscherichia coli 33Met Lys Leu Ile Trp Ser Glu Glu Ser Trp Asp Asp Tyr Leu Tyr Trp 1 5 10 15 Gln Glu Thr Asp Lys Arg Ile Val Lys Lys Ile Asn Glu Leu Ile Lys 20 25 30 Asp Thr Arg Arg Thr Pro Phe Glu Gly Lys Gly Lys Pro Glu Pro Leu 35 40 45 Lys His Asn Leu Ser Gly Phe Trp Ser Arg Arg Ile Thr Glu Glu His 50 55 60 Arg Leu Val Tyr Ala Val Thr Asp Asp Ser Leu Leu Ile Ala Ala Cys 65 70 75 80 Arg Tyr His Tyr 34286PRTEscherichia coli 34Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30 Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp 35 40 45 Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 50 55 60 Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 65 70 75 80 Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 85 90 95 Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 100 105 110 Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser 115 120 125 Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys 130 135 140 Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu 145 150 155 160 Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg 165 170 175 Asp Thr Thr Met Pro Ala Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 180 185 190 Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp 195 200 205 Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 210 215 220 Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser 225 230 235 240 Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile 245 250 255 Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn 260 265 270 Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp 275 280 285 35336DNAEscherichia coliCDS(1)..(336)mazF gene encoding a toxin protein 35atg gta agc cga tac gta ccc gat atg ggc gat ctg att tgg gtt gat 48Met Val Ser Arg Tyr Val Pro Asp Met Gly Asp Leu Ile Trp Val Asp 1 5 10 15 ttt gac ccg aca aaa ggt agc gag caa gct gga cat cgt cca gct gtt 96Phe Asp Pro Thr Lys Gly Ser Glu Gln Ala Gly His Arg Pro Ala Val 20 25 30 gtc ctg agt cct ttc atg tac aac aac aaa aca ggt atg tgt ctg tgt 144Val Leu Ser Pro Phe Met Tyr Asn Asn Lys Thr Gly Met Cys Leu Cys 35 40 45 gtt cct tgt aca acg caa tca aaa gga tat ccg ttc gaa gtt gtt tta 192Val Pro Cys Thr Thr Gln Ser Lys Gly Tyr Pro Phe Glu Val Val Leu 50 55 60 tcc ggt cag gaa cgt gat ggc gta gcg tta gct gat cag gta aaa agt 240Ser Gly Gln Glu Arg Asp Gly Val Ala Leu Ala Asp Gln Val Lys Ser 65 70 75 80 atc gcc tgg cgg gca aga gga gca acg aag aaa gga aca gtt gcc cca 288Ile Ala Trp Arg Ala Arg Gly Ala Thr Lys Lys Gly Thr Val Ala Pro 85 90 95 gag gaa tta caa ctc att aaa gcc aaa att aac gta ctg att ggg tag 336Glu Glu Leu Gln Leu Ile Lys Ala Lys Ile Asn Val Leu Ile Gly 100 105 110 36111PRTEscherichia coli 36Met Val Ser Arg Tyr Val Pro Asp Met Gly Asp Leu Ile Trp Val Asp 1 5 10 15 Phe Asp Pro Thr Lys Gly Ser Glu Gln Ala Gly His Arg Pro Ala Val 20 25 30 Val Leu Ser Pro Phe Met Tyr Asn Asn Lys Thr Gly Met Cys Leu Cys 35 40 45 Val Pro Cys Thr Thr Gln Ser Lys Gly Tyr Pro Phe Glu Val Val Leu 50 55 60 Ser Gly Gln Glu Arg Asp Gly Val Ala Leu Ala Asp Gln Val Lys Ser 65 70 75 80 Ile Ala Trp Arg Ala Arg Gly Ala Thr Lys Lys Gly Thr Val Ala Pro 85 90 95 Glu Glu Leu Gln Leu Ile Lys Ala Lys Ile Asn Val Leu Ile Gly 100 105 110 37249DNAEscherichia coliCDS(1)..(249)mazE gene encoding an antitoxin protein 37atg atc cac agt agc gta aag cgt tgg gga aat tca ccg gcg gtg cgg 48Met Ile His Ser Ser Val Lys Arg Trp Gly Asn Ser Pro Ala Val Arg 1 5 10 15 atc ccg gct acg tta atg cag gcg ctc aat ctg aat att gat gat gaa 96Ile Pro Ala Thr Leu Met Gln Ala Leu Asn Leu Asn Ile Asp Asp Glu 20 25 30 gtg aag att gac ctg gtg gat ggc aaa tta att att gag cca gtg cgt 144Val Lys Ile Asp Leu Val Asp Gly Lys Leu Ile Ile Glu Pro Val Arg 35 40 45 aaa gag ccc gta ttt acg ctt gct gaa ctg gtc aac gac atc acg ccg 192Lys Glu Pro Val Phe Thr Leu Ala Glu Leu Val Asn Asp Ile Thr Pro 50 55 60 gaa aac ctc cac gag aat atc gac tgg gga gag ccg aaa gat aag gaa 240Glu Asn Leu His Glu Asn Ile Asp Trp Gly Glu Pro Lys Asp Lys Glu 65 70 75 80 gtc tgg taa 249Val Trp 3882PRTEscherichia coli 38Met Ile His Ser Ser Val Lys Arg Trp Gly Asn Ser Pro Ala Val Arg 1 5 10 15 Ile Pro Ala Thr Leu Met Gln Ala Leu Asn Leu Asn Ile Asp Asp Glu 20 25 30 Val Lys Ile Asp Leu Val Asp Gly Lys Leu Ile Ile Glu Pro Val Arg 35 40 45 Lys Glu Pro Val Phe Thr Leu Ala Glu Leu Val Asn Asp Ile Thr Pro 50 55 60 Glu Asn Leu His Glu Asn Ile Asp Trp Gly Glu Pro Lys Asp Lys Glu 65 70 75 80 Val Trp

39399DNAShigella sppCDS(1)..(399)yafO gene encoding a toxin 39atg cgg gta ttc aaa aca aaa ctt att cgc ctg caa ctt aca gca gag 48Met Arg Val Phe Lys Thr Lys Leu Ile Arg Leu Gln Leu Thr Ala Glu 1 5 10 15 gaa ctt gat gcg tta acg gcg gat ttt att tcc tat aag cgt gac ggt 96Glu Leu Asp Ala Leu Thr Ala Asp Phe Ile Ser Tyr Lys Arg Asp Gly 20 25 30 gtt ttg cca gat ata ttt ggt cgc gat gca ctc tac gac gac tcc ttt 144Val Leu Pro Asp Ile Phe Gly Arg Asp Ala Leu Tyr Asp Asp Ser Phe 35 40 45 acc tgg cca tta atc aaa ttt gag cga gtt gct cat att cat ctg gca 192Thr Trp Pro Leu Ile Lys Phe Glu Arg Val Ala His Ile His Leu Ala 50 55 60 aat gag aat aat cca ttt ccg cca cag ttg cgc caa ttc agc aga acg 240Asn Glu Asn Asn Pro Phe Pro Pro Gln Leu Arg Gln Phe Ser Arg Thr 65 70 75 80 aat gac gaa gcg cat ttg gta tat tgt cag ggg gcg ttt gat gag caa 288Asn Asp Glu Ala His Leu Val Tyr Cys Gln Gly Ala Phe Asp Glu Gln 85 90 95 gca tgg ttg ctc att gcc att ctg aaa cct gaa cct cat aaa ctg gct 336Ala Trp Leu Leu Ile Ala Ile Leu Lys Pro Glu Pro His Lys Leu Ala 100 105 110 cga gat aac aac caa atg cat aaa att ggg aaa atg gca gaa gcg ttt 384Arg Asp Asn Asn Gln Met His Lys Ile Gly Lys Met Ala Glu Ala Phe 115 120 125 cgc atg cgt ttt tga 399Arg Met Arg Phe 130 40132PRTShigella spp 40Met Arg Val Phe Lys Thr Lys Leu Ile Arg Leu Gln Leu Thr Ala Glu 1 5 10 15 Glu Leu Asp Ala Leu Thr Ala Asp Phe Ile Ser Tyr Lys Arg Asp Gly 20 25 30 Val Leu Pro Asp Ile Phe Gly Arg Asp Ala Leu Tyr Asp Asp Ser Phe 35 40 45 Thr Trp Pro Leu Ile Lys Phe Glu Arg Val Ala His Ile His Leu Ala 50 55 60 Asn Glu Asn Asn Pro Phe Pro Pro Gln Leu Arg Gln Phe Ser Arg Thr 65 70 75 80 Asn Asp Glu Ala His Leu Val Tyr Cys Gln Gly Ala Phe Asp Glu Gln 85 90 95 Ala Trp Leu Leu Ile Ala Ile Leu Lys Pro Glu Pro His Lys Leu Ala 100 105 110 Arg Asp Asn Asn Gln Met His Lys Ile Gly Lys Met Ala Glu Ala Phe 115 120 125 Arg Met Arg Phe 130 41294DNAshigella sppCDS(1)..(294)yafN gene encoding an antitoxin 41atg cat cga att ctc gct gaa aaa tcg gtc aat atc act gag tta cgt 48Met His Arg Ile Leu Ala Glu Lys Ser Val Asn Ile Thr Glu Leu Arg 1 5 10 15 aaa aac cca gct aaa tac ttt att gat caa ccg gtt gcg gtt ctt tct 96Lys Asn Pro Ala Lys Tyr Phe Ile Asp Gln Pro Val Ala Val Leu Ser 20 25 30 aat aat cgc ccc gca gga tat ctc tta agt gcc agc gca ttc gaa gcg 144Asn Asn Arg Pro Ala Gly Tyr Leu Leu Ser Ala Ser Ala Phe Glu Ala 35 40 45 tta atg gac atg ctt gct gaa caa gag gag aaa aag ccc ata aag gcg 192Leu Met Asp Met Leu Ala Glu Gln Glu Glu Lys Lys Pro Ile Lys Ala 50 55 60 cgc ttc cgt cca agt gct gca aga tta gag gaa att aca cgc cgc gct 240Arg Phe Arg Pro Ser Ala Ala Arg Leu Glu Glu Ile Thr Arg Arg Ala 65 70 75 80 gaa caa tat ctt aat gat atg acg gat gat gat ttc aat gac ttt aag 288Glu Gln Tyr Leu Asn Asp Met Thr Asp Asp Asp Phe Asn Asp Phe Lys 85 90 95 gaa taa 294Glu 4297PRTshigella spp 42Met His Arg Ile Leu Ala Glu Lys Ser Val Asn Ile Thr Glu Leu Arg 1 5 10 15 Lys Asn Pro Ala Lys Tyr Phe Ile Asp Gln Pro Val Ala Val Leu Ser 20 25 30 Asn Asn Arg Pro Ala Gly Tyr Leu Leu Ser Ala Ser Ala Phe Glu Ala 35 40 45 Leu Met Asp Met Leu Ala Glu Gln Glu Glu Lys Lys Pro Ile Lys Ala 50 55 60 Arg Phe Arg Pro Ser Ala Ala Arg Leu Glu Glu Ile Thr Arg Arg Ala 65 70 75 80 Glu Gln Tyr Leu Asn Asp Met Thr Asp Asp Asp Phe Asn Asp Phe Lys 85 90 95 Glu 43288DNAEscherichia coliCDS(1)..(288)Rel E gene encoding a toxin 43atg gcg tat ttt ctg gat ttt gac gag cgg gca cta aag gaa tgg cga 48Met Ala Tyr Phe Leu Asp Phe Asp Glu Arg Ala Leu Lys Glu Trp Arg 1 5 10 15 aag ctg ggc tcg acg gta cgt gaa cag ttg aaa aag aag ctg gtt gaa 96Lys Leu Gly Ser Thr Val Arg Glu Gln Leu Lys Lys Lys Leu Val Glu 20 25 30 gta ctt gag tca ccc cgg att gaa gca aac aag ctc cgt ggt atg cct 144Val Leu Glu Ser Pro Arg Ile Glu Ala Asn Lys Leu Arg Gly Met Pro 35 40 45 gat tgt tac aag att aag ctc cgg tct tca ggc tat cgc ctt gta tac 192Asp Cys Tyr Lys Ile Lys Leu Arg Ser Ser Gly Tyr Arg Leu Val Tyr 50 55 60 cag gtt ata gac gag aaa gtt gtc gtt ttc gtg att tct gtt ggg aaa 240Gln Val Ile Asp Glu Lys Val Val Val Phe Val Ile Ser Val Gly Lys 65 70 75 80 aga gaa cgc tcg gaa gta tat agc gag gcg gtc aaa cgc att ctc tga 288Arg Glu Arg Ser Glu Val Tyr Ser Glu Ala Val Lys Arg Ile Leu 85 90 95 4495PRTEscherichia coli 44Met Ala Tyr Phe Leu Asp Phe Asp Glu Arg Ala Leu Lys Glu Trp Arg 1 5 10 15 Lys Leu Gly Ser Thr Val Arg Glu Gln Leu Lys Lys Lys Leu Val Glu 20 25 30 Val Leu Glu Ser Pro Arg Ile Glu Ala Asn Lys Leu Arg Gly Met Pro 35 40 45 Asp Cys Tyr Lys Ile Lys Leu Arg Ser Ser Gly Tyr Arg Leu Val Tyr 50 55 60 Gln Val Ile Asp Glu Lys Val Val Val Phe Val Ile Ser Val Gly Lys 65 70 75 80 Arg Glu Arg Ser Glu Val Tyr Ser Glu Ala Val Lys Arg Ile Leu 85 90 95 45240DNAEscherichia coliCDS(1)..(240)RelB gene encoding an antitoxin 45atg ggt agc att aac ctg cgt att gac gat gaa ctt aaa gcg cgt tct 48Met Gly Ser Ile Asn Leu Arg Ile Asp Asp Glu Leu Lys Ala Arg Ser 1 5 10 15 tac gcc gcg ctt gaa aaa atg ggt gta act cct tct gaa gcg ctt cgt 96Tyr Ala Ala Leu Glu Lys Met Gly Val Thr Pro Ser Glu Ala Leu Arg 20 25 30 ctc atg ctc gag tat atc gct gac aat gaa cgc ttg ccg ttc aaa cag 144Leu Met Leu Glu Tyr Ile Ala Asp Asn Glu Arg Leu Pro Phe Lys Gln 35 40 45 aca ctc ctg agt gat gaa gat gct gaa ctt gtg gag ata gtg aaa gaa 192Thr Leu Leu Ser Asp Glu Asp Ala Glu Leu Val Glu Ile Val Lys Glu 50 55 60 cgg ctt cgt aat cct aag cca gta cgt gtg acg ctg gat gaa ctc tga 240Arg Leu Arg Asn Pro Lys Pro Val Arg Val Thr Leu Asp Glu Leu 65 70 75 4679PRTEscherichia coli 46Met Gly Ser Ile Asn Leu Arg Ile Asp Asp Glu Leu Lys Ala Arg Ser 1 5 10 15 Tyr Ala Ala Leu Glu Lys Met Gly Val Thr Pro Ser Glu Ala Leu Arg 20 25 30 Leu Met Leu Glu Tyr Ile Ala Asp Asn Glu Arg Leu Pro Phe Lys Gln 35 40 45 Thr Leu Leu Ser Asp Glu Asp Ala Glu Leu Val Glu Ile Val Lys Glu 50 55 60 Arg Leu Arg Asn Pro Lys Pro Val Arg Val Thr Leu Asp Glu Leu 65 70 75 4735DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23119 synthetic promoter 47ttgacagcta gctcagtcct aggtataatg ctagc 354835DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23100 synthetic promoter 48ttgacggcta gctcagtcct aggtacagtg ctagc 354935DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23101 synthetic promoter 49tttacagcta gctcagtcct aggtattatg ctagc 355035DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)Synthetic promoter 50ttgacagcta gctcagtcct aggtactgtg ctagc 355135DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23103 synthetic promoter 51ctgatagcta gctcagtcct agggattatg ctagc 355235DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23104 synthetic promoter 52ttgacagcta gctcagtcct aggtattgtg ctagc 355335DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23105 synthetic promoter 53tttacggcta gctcagtcct aggtactatg ctagc 355435DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23106 synthetic promoter 54tttacggcta gctcagtcct aggtatagtg ctagc 355535DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23107 synthetic promoter 55tttacggcta gctcagccct aggtattatg ctagc 355635DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23108 synthetic promoter 56ctgacagcta gctcagtcct aggtataatg ctagc 355735DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23109 synthetic promoter 57tttacagcta gctcagtcct agggactgtg ctagc 355835DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23110 synthetic promoter 58tttacggcta gctcagtcct aggtacaatg ctagc 355935DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23111 synthetic promoter 59ttgacggcta gctcagtcct aggtatagtg ctagc 356035DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23112 synthetic promoter 60ctgatagcta gctcagtcct agggattatg ctagc 356135DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23113 synthetic promoter 61ctgatggcta gctcagtcct agggattatg ctagc 356235DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23114 synthetic promoter 62tttatggcta gctcagtcct aggtacaatg ctagc 356335DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23115 synthetic promoter 63tttatagcta gctcagccct tggtacaatg ctagc 356435DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23116 synthetic promoter 64ttgacagcta gctcagtcct agggactatg ctagc 356535DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidepromoter(1)..(35)J23117 synthetic promoter 65ttgacagcta gctcagtcct agggattgtg ctagc 3566355DNAEscherichia colipromoter(1)..(355)pBAD promoter 66acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg 60ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa 120aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta 180taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat 240agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac 300tgtttctcca tacccgtttt tttgggctag cgaattcgag ctcgaggagg aaggt 35567720DNAEscherichia coliCDS(1)..(720)FadR gene encoding a fatty acid sensor 67atg gtc att aag gcg caa agc ccg gcg ggt ttc gcg gaa gag tac att 48Met Val Ile Lys Ala Gln Ser Pro Ala Gly Phe Ala Glu Glu Tyr Ile 1 5 10 15 att gaa agt atc tgg aat aac cgc ttc cct ccc ggg act att ttg ccc 96Ile Glu Ser Ile Trp Asn Asn Arg Phe Pro Pro Gly Thr Ile Leu Pro 20 25 30 gca gaa cgt gaa ctt tca gaa tta att ggc gta acg cgt act acg tta 144Ala Glu Arg Glu Leu Ser Glu Leu Ile Gly Val Thr Arg Thr Thr Leu 35 40 45 cgt gaa gtg tta cag cgt ctg gca cga gat ggc tgg ttg acc att caa 192Arg Glu Val Leu Gln Arg Leu Ala Arg Asp Gly Trp Leu Thr Ile Gln 50 55 60 cat ggc aag ccg acg aag gtg aat aat ttc tgg gaa act tcc ggt tta 240His Gly Lys Pro Thr Lys Val Asn Asn Phe Trp Glu Thr Ser Gly Leu 65 70 75 80 aat atc ctt gaa aca ctg gcg cga ctg gat cac gaa agt gtg ccg cag 288Asn Ile Leu Glu Thr Leu Ala Arg Leu Asp His Glu Ser Val Pro Gln 85 90 95 ctt att gat aat ttg ctg tcg gtg cgt acc aat att tcc act att ttt 336Leu Ile Asp Asn Leu Leu Ser Val Arg Thr Asn Ile Ser Thr Ile Phe 100 105 110 att cgc acc gcg ttt cgt cag cat ccc gat aaa gcg cag gaa gtg ctg 384Ile Arg Thr Ala Phe Arg Gln His Pro Asp Lys Ala Gln Glu Val Leu 115 120 125 gct acc gct aat gaa gtg gcc gat cac gcc gat gcc ttt gcc gag ctg 432Ala Thr Ala Asn Glu Val Ala Asp His Ala Asp Ala Phe Ala Glu Leu 130 135 140 gat tac aac ata ttc cgc ggc ctg gcg ttt gct tcc ggc aac ccg att 480Asp Tyr Asn Ile Phe Arg Gly Leu Ala Phe Ala Ser Gly Asn Pro Ile 145 150 155 160 tac ggt ctg att ctt aac ggg atg aaa ggg ctg tat acg cgt att ggt 528Tyr Gly Leu Ile Leu Asn Gly Met Lys Gly Leu Tyr Thr Arg Ile Gly 165 170 175 cgt cac tat ttc gcc aat ccg gaa gcg cgc agt ctg gcg ctg ggc ttc 576Arg His Tyr Phe Ala Asn Pro Glu Ala Arg Ser Leu Ala Leu Gly Phe 180 185 190 tac cac aaa ctg tcg gcg ttg tgc agt gaa ggc gcg cac gat cag gtg 624Tyr His Lys Leu Ser Ala Leu Cys Ser Glu Gly Ala His Asp Gln Val 195 200 205 tac gaa aca gtg cgt cgc tat ggg cat gag agt ggc gag att tgg cac 672Tyr Glu Thr Val Arg Arg Tyr Gly His Glu Ser Gly Glu Ile Trp His 210 215 220 cgg atg cag aaa aat ctg ccg ggt gat tta gcc att cag ggg cga taa 720Arg Met Gln Lys Asn Leu Pro Gly Asp Leu Ala Ile Gln Gly Arg 225 230 235 68239PRTEscherichia coli 68Met Val Ile Lys Ala Gln Ser Pro Ala Gly Phe Ala Glu Glu Tyr Ile 1 5 10 15 Ile Glu Ser Ile Trp Asn Asn Arg Phe Pro Pro Gly Thr Ile Leu Pro 20 25 30 Ala Glu Arg Glu Leu Ser Glu Leu Ile Gly Val Thr Arg Thr Thr Leu 35 40 45 Arg Glu Val Leu Gln Arg Leu Ala Arg Asp Gly Trp Leu Thr Ile Gln 50 55 60 His Gly Lys Pro Thr Lys Val Asn Asn Phe Trp Glu Thr Ser Gly Leu 65 70 75 80 Asn Ile Leu Glu Thr Leu Ala Arg Leu Asp His Glu Ser Val Pro Gln 85 90 95 Leu Ile Asp Asn Leu Leu Ser Val Arg Thr Asn Ile Ser Thr Ile Phe 100 105 110 Ile Arg Thr Ala Phe Arg Gln His

Pro Asp Lys Ala Gln Glu Val Leu 115 120 125 Ala Thr Ala Asn Glu Val Ala Asp His Ala Asp Ala Phe Ala Glu Leu 130 135 140 Asp Tyr Asn Ile Phe Arg Gly Leu Ala Phe Ala Ser Gly Asn Pro Ile 145 150 155 160 Tyr Gly Leu Ile Leu Asn Gly Met Lys Gly Leu Tyr Thr Arg Ile Gly 165 170 175 Arg His Tyr Phe Ala Asn Pro Glu Ala Arg Ser Leu Ala Leu Gly Phe 180 185 190 Tyr His Lys Leu Ser Ala Leu Cys Ser Glu Gly Ala His Asp Gln Val 195 200 205 Tyr Glu Thr Val Arg Arg Tyr Gly His Glu Ser Gly Glu Ile Trp His 210 215 220 Arg Met Gln Lys Asn Leu Pro Gly Asp Leu Ala Ile Gln Gly Arg 225 230 235 69240DNAEscherichia colimisc_feature(1)..(240)btuB leader (adenosylcobalamin) riboswtich 69gccggtcctg tgagttaata gggaatccag tgcgaatctg gagctgacgc gcagcggtaa 60ggaaaggtgc gatgattgcg ttatgcggac actgccattc ggtgggaagt catcatctct 120tagtatctta gatacccctc caagcccgaa gacctgccgg ccaacgtcgc atctggttct 180catcatcgcg taatattgat gaaacctgcg gcatccttct tctattgtgg atgctttaca 24070279DNAEscherichia colimisc_feature(1)..(279)tc3 (tetracycline) riboswitch 70ccgcggccaa ttatctactt aaggcctaaa acataccaga tcgccacccg cgctttaatc 60tggagaggtg aagaatacga ccacctaggc cttaagaacc ggtaaaacat accagatcgc 120cacccgcgct ttaatctgga gaggtgaaga atacgaccac ctaccggtca acaacaacaa 180caacaacaac aacaactcga ggcctaaaac ataccagatc gccacccgcg ctttaatctg 240gagaggtgaa gaatacgacc acctaggcct cgagaacat 279711260DNAEscherichia coliCDS(1)..(1260)xylR gene encoding a xyl repressor sensor protein 71atg caa aac aac ttg ttg ttc ttg tgc aag aag tac tac aac atc atc 48Met Gln Asn Asn Leu Leu Phe Leu Cys Lys Lys Tyr Tyr Asn Ile Ile 1 5 10 15 ttg aga aga aac atc aag ttg gtc acc gat aag tac acc atc aga gaa 96Leu Arg Arg Asn Ile Lys Leu Val Thr Asp Lys Tyr Thr Ile Arg Glu 20 25 30 atg aac gaa aga ttg gtc ttg gaa caa atc atc aag aac ggt cca att 144Met Asn Glu Arg Leu Val Leu Glu Gln Ile Ile Lys Asn Gly Pro Ile 35 40 45 tcc aga gct tct att gct tct act atc ggt ttg aac aag gct acc att 192Ser Arg Ala Ser Ile Ala Ser Thr Ile Gly Leu Asn Lys Ala Thr Ile 50 55 60 tct gcc att acc aag aag ttg att gac gaa tct ttg gtc cac gaa att 240Ser Ala Ile Thr Lys Lys Leu Ile Asp Glu Ser Leu Val His Glu Ile 65 70 75 80 ggt att ggt aac tct act cat tct ggt ggt aga aag cca atc ttg ttg 288Gly Ile Gly Asn Ser Thr His Ser Gly Gly Arg Lys Pro Ile Leu Leu 85 90 95 gtt ttt aac aag tgc gcc ggt atc tct ttg tct atg gat att ggt tac 336Val Phe Asn Lys Cys Ala Gly Ile Ser Leu Ser Met Asp Ile Gly Tyr 100 105 110 gac tac atc ttc tcc tca ttg tct tat ttg gac ggt act atc atc aac 384Asp Tyr Ile Phe Ser Ser Leu Ser Tyr Leu Asp Gly Thr Ile Ile Asn 115 120 125 tcc aaa aag ttg acc gat atc caa gtc tcc aag gat aac gtc att caa 432Ser Lys Lys Leu Thr Asp Ile Gln Val Ser Lys Asp Asn Val Ile Gln 130 135 140 ttg atc gac gaa att atc aac tcc tac aac att tcc aag atc gac act 480Leu Ile Asp Glu Ile Ile Asn Ser Tyr Asn Ile Ser Lys Ile Asp Thr 145 150 155 160 cca tac aag gtt att ggt ttg acc ttg gcc att cat ggt att acc tgt 528Pro Tyr Lys Val Ile Gly Leu Thr Leu Ala Ile His Gly Ile Thr Cys 165 170 175 gaa aac aag gtt ttg ttc acc cct tac tac aac ttg aac gaa atc gac 576Glu Asn Lys Val Leu Phe Thr Pro Tyr Tyr Asn Leu Asn Glu Ile Asp 180 185 190 ttg tac tcc atc ttg tcc aag aaa tac gat ttc cca atc cac att gaa 624Leu Tyr Ser Ile Leu Ser Lys Lys Tyr Asp Phe Pro Ile His Ile Glu 195 200 205 aac gaa gct aac ttg act gct ttg gct gaa aac act ttc tct acc gtt 672Asn Glu Ala Asn Leu Thr Ala Leu Ala Glu Asn Thr Phe Ser Thr Val 210 215 220 cat aac tcc ttg ttg tct ttg tcc atc cat tct ggt ttt ggt tcc ggt 720His Asn Ser Leu Leu Ser Leu Ser Ile His Ser Gly Phe Gly Ser Gly 225 230 235 240 atc att atc aac aac aaa tta tac tcc ggt aga aac ggt atg tcc ggt 768Ile Ile Ile Asn Asn Lys Leu Tyr Ser Gly Arg Asn Gly Met Ser Gly 245 250 255 gaa att ggt cat acc att att atg cca aac ggt aaa ttg tgt cca tgc 816Glu Ile Gly His Thr Ile Ile Met Pro Asn Gly Lys Leu Cys Pro Cys 260 265 270 ggt aat aga ggt tgt ttg gaa caa tac tgc tcc gaa aag aag gtc ttt 864Gly Asn Arg Gly Cys Leu Glu Gln Tyr Cys Ser Glu Lys Lys Val Phe 275 280 285 gaa caa ttg tcc tcc ttg gaa aac atc cca aag atc gat tct gat atc 912Glu Gln Leu Ser Ser Leu Glu Asn Ile Pro Lys Ile Asp Ser Asp Ile 290 295 300 gtc aag caa ttg tac tac gaa gat aat caa aac gcc aag aag gtc atc 960Val Lys Gln Leu Tyr Tyr Glu Asp Asn Gln Asn Ala Lys Lys Val Ile 305 310 315 320 cac gaa ttt tgt tct tac ttg acc att gcc att aac aac gct att act 1008His Glu Phe Cys Ser Tyr Leu Thr Ile Ala Ile Asn Asn Ala Ile Thr 325 330 335 act tac gcc cca gaa atc atc tac ttg aac tcc caa att atc tct gac 1056Thr Tyr Ala Pro Glu Ile Ile Tyr Leu Asn Ser Gln Ile Ile Ser Asp 340 345 350 att cca gaa atc ttg caa atc acc aag gac atg ttg gtc agt tct ttc 1104Ile Pro Glu Ile Leu Gln Ile Thr Lys Asp Met Leu Val Ser Ser Phe 355 360 365 aac aag ggt atc aac atc gaa atc tcc agt ttg ggt tcc gaa gct tca 1152Asn Lys Gly Ile Asn Ile Glu Ile Ser Ser Leu Gly Ser Glu Ala Ser 370 375 380 tta tat ggt ggt tct gct gtc aac atc aag agt ttc ttg aac atc caa 1200Leu Tyr Gly Gly Ser Ala Val Asn Ile Lys Ser Phe Leu Asn Ile Gln 385 390 395 400 aac ttg acc ttg att aac gaa ata ggt tcc cca aag aag aag aga aag 1248Asn Leu Thr Leu Ile Asn Glu Ile Gly Ser Pro Lys Lys Lys Arg Lys 405 410 415 gtt acc aga tga 1260Val Thr Arg 72419PRTEscherichia coli 72Met Gln Asn Asn Leu Leu Phe Leu Cys Lys Lys Tyr Tyr Asn Ile Ile 1 5 10 15 Leu Arg Arg Asn Ile Lys Leu Val Thr Asp Lys Tyr Thr Ile Arg Glu 20 25 30 Met Asn Glu Arg Leu Val Leu Glu Gln Ile Ile Lys Asn Gly Pro Ile 35 40 45 Ser Arg Ala Ser Ile Ala Ser Thr Ile Gly Leu Asn Lys Ala Thr Ile 50 55 60 Ser Ala Ile Thr Lys Lys Leu Ile Asp Glu Ser Leu Val His Glu Ile 65 70 75 80 Gly Ile Gly Asn Ser Thr His Ser Gly Gly Arg Lys Pro Ile Leu Leu 85 90 95 Val Phe Asn Lys Cys Ala Gly Ile Ser Leu Ser Met Asp Ile Gly Tyr 100 105 110 Asp Tyr Ile Phe Ser Ser Leu Ser Tyr Leu Asp Gly Thr Ile Ile Asn 115 120 125 Ser Lys Lys Leu Thr Asp Ile Gln Val Ser Lys Asp Asn Val Ile Gln 130 135 140 Leu Ile Asp Glu Ile Ile Asn Ser Tyr Asn Ile Ser Lys Ile Asp Thr 145 150 155 160 Pro Tyr Lys Val Ile Gly Leu Thr Leu Ala Ile His Gly Ile Thr Cys 165 170 175 Glu Asn Lys Val Leu Phe Thr Pro Tyr Tyr Asn Leu Asn Glu Ile Asp 180 185 190 Leu Tyr Ser Ile Leu Ser Lys Lys Tyr Asp Phe Pro Ile His Ile Glu 195 200 205 Asn Glu Ala Asn Leu Thr Ala Leu Ala Glu Asn Thr Phe Ser Thr Val 210 215 220 His Asn Ser Leu Leu Ser Leu Ser Ile His Ser Gly Phe Gly Ser Gly 225 230 235 240 Ile Ile Ile Asn Asn Lys Leu Tyr Ser Gly Arg Asn Gly Met Ser Gly 245 250 255 Glu Ile Gly His Thr Ile Ile Met Pro Asn Gly Lys Leu Cys Pro Cys 260 265 270 Gly Asn Arg Gly Cys Leu Glu Gln Tyr Cys Ser Glu Lys Lys Val Phe 275 280 285 Glu Gln Leu Ser Ser Leu Glu Asn Ile Pro Lys Ile Asp Ser Asp Ile 290 295 300 Val Lys Gln Leu Tyr Tyr Glu Asp Asn Gln Asn Ala Lys Lys Val Ile 305 310 315 320 His Glu Phe Cys Ser Tyr Leu Thr Ile Ala Ile Asn Asn Ala Ile Thr 325 330 335 Thr Tyr Ala Pro Glu Ile Ile Tyr Leu Asn Ser Gln Ile Ile Ser Asp 340 345 350 Ile Pro Glu Ile Leu Gln Ile Thr Lys Asp Met Leu Val Ser Ser Phe 355 360 365 Asn Lys Gly Ile Asn Ile Glu Ile Ser Ser Leu Gly Ser Glu Ala Ser 370 375 380 Leu Tyr Gly Gly Ser Ala Val Asn Ile Lys Ser Phe Leu Asn Ile Gln 385 390 395 400 Asn Leu Thr Leu Ile Asn Glu Ile Gly Ser Pro Lys Lys Lys Arg Lys 405 410 415 Val Thr Arg 73351DNASaccharomyces cerevisiaepromoter(1)..(351)xylR-xylose responsive promoter 73atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat cgccgtacca 60cttcaaaaca cccaagcaca gcatactaaa tttcccctct ttcttcctct agggtgtcgt 120taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc 180gtcgaaaaag gcaataaaaa tttttatata aataagttag tttaatatac taacaaacta 240atactttcaa cattttcagt ttgtattact tcttattcaa atgtcataaa agtatcaaca 300aaaaattgtt aatatacctc tatactttaa cgtcaaggag aaaaaactat a 35174133DNAEscherichia colimisc_feature(1)..(133)ThiMribo-wt (TPP) riboswitch 74aaccaaacga ctcggggtgc ccttctgcgt gaaggctgag aaatacccgt atcacctgat 60ctggataatg ccagcgtagg gaagtcacgg accaccaggt cattgcttct tcacgttatg 120gcaggagcaa act 133751260DNAEscherichia coliCDS(1)..(1260)MurA gene encodes UDP-N-acetylglucosamine enolpyruvyl transferase 75atg gat aaa ttt cgt gtt cag ggg cca acg aag ctc cag ggc gaa gtc 48Met Asp Lys Phe Arg Val Gln Gly Pro Thr Lys Leu Gln Gly Glu Val 1 5 10 15 aca att tcc ggc gct aaa aat gct gct ctg cct atc ctt ttt gcc gca 96Thr Ile Ser Gly Ala Lys Asn Ala Ala Leu Pro Ile Leu Phe Ala Ala 20 25 30 cta ctg gcg gaa gaa ccg gta gag atc cag aac gtc ccg aaa ctg aaa 144Leu Leu Ala Glu Glu Pro Val Glu Ile Gln Asn Val Pro Lys Leu Lys 35 40 45 gac gtc gat aca tca atg aag ctg cta agc cag ctg ggt gcg aaa gta 192Asp Val Asp Thr Ser Met Lys Leu Leu Ser Gln Leu Gly Ala Lys Val 50 55 60 gaa cgt aat ggt tct gtg cat att gat gcc cgc gac gtt aat gta ttc 240Glu Arg Asn Gly Ser Val His Ile Asp Ala Arg Asp Val Asn Val Phe 65 70 75 80 tgc gca cct tac gat ctg gtt aaa acc atg cgt gct tct atc tgg gcg 288Cys Ala Pro Tyr Asp Leu Val Lys Thr Met Arg Ala Ser Ile Trp Ala 85 90 95 ctg ggg ccg ctg gta gcg cgc ttt ggt cag ggg caa gtt tca cta cct 336Leu Gly Pro Leu Val Ala Arg Phe Gly Gln Gly Gln Val Ser Leu Pro 100 105 110 ggc ggt tgt acg atc ggt gcg cgt ccg gtt gat cta cac att tct ggc 384Gly Gly Cys Thr Ile Gly Ala Arg Pro Val Asp Leu His Ile Ser Gly 115 120 125 ctc gaa caa tta ggc gcg acc atc aaa ctg gaa gaa ggt tac gtt aaa 432Leu Glu Gln Leu Gly Ala Thr Ile Lys Leu Glu Glu Gly Tyr Val Lys 130 135 140 gct tcc gtc gat ggt cgt ttg aaa ggt gca cat atc gtg atg gat aaa 480Ala Ser Val Asp Gly Arg Leu Lys Gly Ala His Ile Val Met Asp Lys 145 150 155 160 gtc agc gtt ggc gca acg gtg acc atc atg tgt gct gca acc ctg gcg 528Val Ser Val Gly Ala Thr Val Thr Ile Met Cys Ala Ala Thr Leu Ala 165 170 175 gaa ggc acc acg att att gaa aac gca gcg cgt gaa ccg gaa atc gtc 576Glu Gly Thr Thr Ile Ile Glu Asn Ala Ala Arg Glu Pro Glu Ile Val 180 185 190 gat acc gcg aac ttc ctg att acg ctg ggt gcg aaa att agc ggt cag 624Asp Thr Ala Asn Phe Leu Ile Thr Leu Gly Ala Lys Ile Ser Gly Gln 195 200 205 ggc acc gat cgt atc gtc atc gaa ggt gtg gaa cgt tta ggc ggc ggt 672Gly Thr Asp Arg Ile Val Ile Glu Gly Val Glu Arg Leu Gly Gly Gly 210 215 220 gtc tat cgc gtt ctg ccg gat cgt atc gaa acc ggt act ttc ctg gtg 720Val Tyr Arg Val Leu Pro Asp Arg Ile Glu Thr Gly Thr Phe Leu Val 225 230 235 240 gcg gcg gcg att tct cgc ggc aaa att atc tgc cgt aac gcg cag cca 768Ala Ala Ala Ile Ser Arg Gly Lys Ile Ile Cys Arg Asn Ala Gln Pro 245 250 255 gat act ctc gac gcc gtg ctg gcg aaa ctg cgt gac gct gga gcg gac 816Asp Thr Leu Asp Ala Val Leu Ala Lys Leu Arg Asp Ala Gly Ala Asp 260 265 270 atc gaa gtc ggc gaa gac tgg att agc ctg gat atg cat ggc aaa cgt 864Ile Glu Val Gly Glu Asp Trp Ile Ser Leu Asp Met His Gly Lys Arg 275 280 285 ccg aag gct gtt aac gta cgt acc gcg ccg cat ccg gca ttc ccg acc 912Pro Lys Ala Val Asn Val Arg Thr Ala Pro His Pro Ala Phe Pro Thr 290 295 300 gat atg cag gcc cag ttc acg ctg ttg aac ctg gtg gca gaa ggg acc 960Asp Met Gln Ala Gln Phe Thr Leu Leu Asn Leu Val Ala Glu Gly Thr 305 310 315 320 ggg ttt atc acc gaa acg gtc ttt gaa aac cgc ttt atg cat gtg cca 1008Gly Phe Ile Thr Glu Thr Val Phe Glu Asn Arg Phe Met His Val Pro 325 330 335 gag ctg agc cgt atg ggc gcg cac gcc gaa atc gaa agc aat acc gtt 1056Glu Leu Ser Arg Met Gly Ala His Ala Glu Ile Glu Ser Asn Thr Val 340 345 350 att tgt cac ggt gtt gaa aaa ctt tct ggc gca cag gtt atg gca acc 1104Ile Cys His Gly Val Glu Lys Leu Ser Gly Ala Gln Val Met Ala Thr 355 360 365 gat ctg cgt gca tca gca agc ctg gtg ctg gct ggc tgt att gcg gaa 1152Asp Leu Arg Ala Ser Ala Ser Leu Val Leu Ala Gly Cys Ile Ala Glu 370 375 380 ggg acg acg gtg gtt gat cgt att tat cac atc gat cgt ggc tac gaa 1200Gly Thr Thr Val Val Asp Arg Ile Tyr His Ile Asp Arg Gly Tyr Glu 385 390 395 400 cgc att gaa gac aaa ctg cgc gct tta ggt gca aat att gag cgt gtg 1248Arg Ile Glu Asp Lys Leu Arg Ala Leu Gly Ala Asn Ile Glu Arg Val 405 410 415 aaa ggc gaa taa 1260Lys Gly Glu 76419PRTEscherichia coli 76Met Asp Lys Phe Arg Val Gln Gly Pro Thr Lys Leu Gln Gly Glu Val 1 5 10 15 Thr Ile Ser Gly Ala Lys Asn Ala Ala Leu Pro Ile Leu Phe Ala Ala 20 25 30 Leu Leu Ala Glu Glu Pro Val Glu Ile Gln Asn Val Pro Lys Leu Lys 35 40 45 Asp Val Asp Thr Ser Met Lys Leu Leu Ser Gln Leu Gly Ala Lys Val 50 55

60 Glu Arg Asn Gly Ser Val His Ile Asp Ala Arg Asp Val Asn Val Phe 65 70 75 80 Cys Ala Pro Tyr Asp Leu Val Lys Thr Met Arg Ala Ser Ile Trp Ala 85 90 95 Leu Gly Pro Leu Val Ala Arg Phe Gly Gln Gly Gln Val Ser Leu Pro 100 105 110 Gly Gly Cys Thr Ile Gly Ala Arg Pro Val Asp Leu His Ile Ser Gly 115 120 125 Leu Glu Gln Leu Gly Ala Thr Ile Lys Leu Glu Glu Gly Tyr Val Lys 130 135 140 Ala Ser Val Asp Gly Arg Leu Lys Gly Ala His Ile Val Met Asp Lys 145 150 155 160 Val Ser Val Gly Ala Thr Val Thr Ile Met Cys Ala Ala Thr Leu Ala 165 170 175 Glu Gly Thr Thr Ile Ile Glu Asn Ala Ala Arg Glu Pro Glu Ile Val 180 185 190 Asp Thr Ala Asn Phe Leu Ile Thr Leu Gly Ala Lys Ile Ser Gly Gln 195 200 205 Gly Thr Asp Arg Ile Val Ile Glu Gly Val Glu Arg Leu Gly Gly Gly 210 215 220 Val Tyr Arg Val Leu Pro Asp Arg Ile Glu Thr Gly Thr Phe Leu Val 225 230 235 240 Ala Ala Ala Ile Ser Arg Gly Lys Ile Ile Cys Arg Asn Ala Gln Pro 245 250 255 Asp Thr Leu Asp Ala Val Leu Ala Lys Leu Arg Asp Ala Gly Ala Asp 260 265 270 Ile Glu Val Gly Glu Asp Trp Ile Ser Leu Asp Met His Gly Lys Arg 275 280 285 Pro Lys Ala Val Asn Val Arg Thr Ala Pro His Pro Ala Phe Pro Thr 290 295 300 Asp Met Gln Ala Gln Phe Thr Leu Leu Asn Leu Val Ala Glu Gly Thr 305 310 315 320 Gly Phe Ile Thr Glu Thr Val Phe Glu Asn Arg Phe Met His Val Pro 325 330 335 Glu Leu Ser Arg Met Gly Ala His Ala Glu Ile Glu Ser Asn Thr Val 340 345 350 Ile Cys His Gly Val Glu Lys Leu Ser Gly Ala Gln Val Met Ala Thr 355 360 365 Asp Leu Arg Ala Ser Ala Ser Leu Val Leu Ala Gly Cys Ile Ala Glu 370 375 380 Gly Thr Thr Val Val Asp Arg Ile Tyr His Ile Asp Arg Gly Tyr Glu 385 390 395 400 Arg Ile Glu Asp Lys Leu Arg Ala Leu Gly Ala Asn Ile Glu Arg Val 405 410 415 Lys Gly Glu 771083DNAEscherichia coliCDS(1)..(1083)mraY gene encodes UDP-MurNAc-pentapeptide phosphotransferase 77atg tta gtt tgg ctg gcc gaa cat ttg gtc aaa tat tat tcc ggc ttt 48Met Leu Val Trp Leu Ala Glu His Leu Val Lys Tyr Tyr Ser Gly Phe 1 5 10 15 aac gtc ttt tcc tat ctg acg ttt cgc gcc atc gtc agc ctg ctg acc 96Asn Val Phe Ser Tyr Leu Thr Phe Arg Ala Ile Val Ser Leu Leu Thr 20 25 30 gcg ctg ttc atc tca ttg tgg atg ggc ccg cgt atg att gct cat ttg 144Ala Leu Phe Ile Ser Leu Trp Met Gly Pro Arg Met Ile Ala His Leu 35 40 45 caa aaa ctt tcc ttt ggt cag gtg gtg cgt aac gac ggt cct gaa tca 192Gln Lys Leu Ser Phe Gly Gln Val Val Arg Asn Asp Gly Pro Glu Ser 50 55 60 cac ttc agc aag cgc ggt acg ccg acc atg ggc ggg att atg atc ctg 240His Phe Ser Lys Arg Gly Thr Pro Thr Met Gly Gly Ile Met Ile Leu 65 70 75 80 acg gcg att gtg atc tcc gta ctg ctg tgg gct tac ccg tcc aat ccg 288Thr Ala Ile Val Ile Ser Val Leu Leu Trp Ala Tyr Pro Ser Asn Pro 85 90 95 tac gtc tgg tgc gtg ttg gtg gtg ctg gta ggt tac ggt gtt att ggc 336Tyr Val Trp Cys Val Leu Val Val Leu Val Gly Tyr Gly Val Ile Gly 100 105 110 ttt gtt gat gat tat cgc aaa gtg gtg cgt aaa gac acc aaa ggg ttg 384Phe Val Asp Asp Tyr Arg Lys Val Val Arg Lys Asp Thr Lys Gly Leu 115 120 125 atc gct cgt tgg aag tat ttc tgg atg tcg gtc att gcg ctg ggt gtc 432Ile Ala Arg Trp Lys Tyr Phe Trp Met Ser Val Ile Ala Leu Gly Val 130 135 140 gcc ttc gcc ctg tac ctt gcc ggc aaa gac acg ccc gca acg cag ctg 480Ala Phe Ala Leu Tyr Leu Ala Gly Lys Asp Thr Pro Ala Thr Gln Leu 145 150 155 160 gtg gtc cca ttc ttt aaa gat gtg atg ccg cag ctg ggg ctg ttc tac 528Val Val Pro Phe Phe Lys Asp Val Met Pro Gln Leu Gly Leu Phe Tyr 165 170 175 att ctg ctg gct tac ttc gtc att gtg ggt act ggc aac gcg gta aac 576Ile Leu Leu Ala Tyr Phe Val Ile Val Gly Thr Gly Asn Ala Val Asn 180 185 190 ctg acc gat ggt ctc gac ggc ctg gca att atg ccg acc gta ttt gtc 624Leu Thr Asp Gly Leu Asp Gly Leu Ala Ile Met Pro Thr Val Phe Val 195 200 205 gcc ggt ggt ttt gcg ctg gtg gcg tgg gcg acc ggc aat atg aac ttt 672Ala Gly Gly Phe Ala Leu Val Ala Trp Ala Thr Gly Asn Met Asn Phe 210 215 220 gcc agc tac ttg cat ata ccg tat ctg cga cac gcc ggg gaa ctg gtt 720Ala Ser Tyr Leu His Ile Pro Tyr Leu Arg His Ala Gly Glu Leu Val 225 230 235 240 att gtc tgt acc gcg ata gtc ggg gca gga ctg ggc ttc ctg tgg ttt 768Ile Val Cys Thr Ala Ile Val Gly Ala Gly Leu Gly Phe Leu Trp Phe 245 250 255 aac acc tat ccg gcg cag gtc ttt atg ggc gat gta ggt tcg ctg gcg 816Asn Thr Tyr Pro Ala Gln Val Phe Met Gly Asp Val Gly Ser Leu Ala 260 265 270 tta ggt ggt gcg tta ggc att atc gcc gta ctg cta cgt cag gaa ttc 864Leu Gly Gly Ala Leu Gly Ile Ile Ala Val Leu Leu Arg Gln Glu Phe 275 280 285 ctg ctg gtg att atg ggg ggc gtg ttc gtg gta gaa acg ctt tct gtc 912Leu Leu Val Ile Met Gly Gly Val Phe Val Val Glu Thr Leu Ser Val 290 295 300 atc ctg cag gtc ggc tcc ttt aaa ctg cgc gga caa cgt att ttc cgc 960Ile Leu Gln Val Gly Ser Phe Lys Leu Arg Gly Gln Arg Ile Phe Arg 305 310 315 320 atg gca ccg att cat cac cac tat gaa ctg aaa ggc tgg ccg gaa ccg 1008Met Ala Pro Ile His His His Tyr Glu Leu Lys Gly Trp Pro Glu Pro 325 330 335 cgc gtc att gtg cgt ttc tgg att att tcg ctg atg ctg gtt ctg att 1056Arg Val Ile Val Arg Phe Trp Ile Ile Ser Leu Met Leu Val Leu Ile 340 345 350 ggt ctg gca acg ctg aag gta cgt taa 1083Gly Leu Ala Thr Leu Lys Val Arg 355 360 78360PRTEscherichia coli 78Met Leu Val Trp Leu Ala Glu His Leu Val Lys Tyr Tyr Ser Gly Phe 1 5 10 15 Asn Val Phe Ser Tyr Leu Thr Phe Arg Ala Ile Val Ser Leu Leu Thr 20 25 30 Ala Leu Phe Ile Ser Leu Trp Met Gly Pro Arg Met Ile Ala His Leu 35 40 45 Gln Lys Leu Ser Phe Gly Gln Val Val Arg Asn Asp Gly Pro Glu Ser 50 55 60 His Phe Ser Lys Arg Gly Thr Pro Thr Met Gly Gly Ile Met Ile Leu 65 70 75 80 Thr Ala Ile Val Ile Ser Val Leu Leu Trp Ala Tyr Pro Ser Asn Pro 85 90 95 Tyr Val Trp Cys Val Leu Val Val Leu Val Gly Tyr Gly Val Ile Gly 100 105 110 Phe Val Asp Asp Tyr Arg Lys Val Val Arg Lys Asp Thr Lys Gly Leu 115 120 125 Ile Ala Arg Trp Lys Tyr Phe Trp Met Ser Val Ile Ala Leu Gly Val 130 135 140 Ala Phe Ala Leu Tyr Leu Ala Gly Lys Asp Thr Pro Ala Thr Gln Leu 145 150 155 160 Val Val Pro Phe Phe Lys Asp Val Met Pro Gln Leu Gly Leu Phe Tyr 165 170 175 Ile Leu Leu Ala Tyr Phe Val Ile Val Gly Thr Gly Asn Ala Val Asn 180 185 190 Leu Thr Asp Gly Leu Asp Gly Leu Ala Ile Met Pro Thr Val Phe Val 195 200 205 Ala Gly Gly Phe Ala Leu Val Ala Trp Ala Thr Gly Asn Met Asn Phe 210 215 220 Ala Ser Tyr Leu His Ile Pro Tyr Leu Arg His Ala Gly Glu Leu Val 225 230 235 240 Ile Val Cys Thr Ala Ile Val Gly Ala Gly Leu Gly Phe Leu Trp Phe 245 250 255 Asn Thr Tyr Pro Ala Gln Val Phe Met Gly Asp Val Gly Ser Leu Ala 260 265 270 Leu Gly Gly Ala Leu Gly Ile Ile Ala Val Leu Leu Arg Gln Glu Phe 275 280 285 Leu Leu Val Ile Met Gly Gly Val Phe Val Val Glu Thr Leu Ser Val 290 295 300 Ile Leu Gln Val Gly Ser Phe Lys Leu Arg Gly Gln Arg Ile Phe Arg 305 310 315 320 Met Ala Pro Ile His His His Tyr Glu Leu Lys Gly Trp Pro Glu Pro 325 330 335 Arg Val Ile Val Arg Phe Trp Ile Ile Ser Leu Met Leu Val Leu Ile 340 345 350 Gly Leu Ala Thr Leu Lys Val Arg 355 360 791338DNAEscherichia coliCDS(1)..(1338)glmM encodes a phosphoglucosamine mutase 79atg agt aat cgt aaa tat ttc ggt acc gat ggg att cgt ggt cgt gta 48Met Ser Asn Arg Lys Tyr Phe Gly Thr Asp Gly Ile Arg Gly Arg Val 1 5 10 15 ggg gat gcg ccg atc aca cct gat ttt gtg ctt aag ctg ggt tgg gcc 96Gly Asp Ala Pro Ile Thr Pro Asp Phe Val Leu Lys Leu Gly Trp Ala 20 25 30 gcg ggt aaa gtg ctg gcg cgc cac ggc tcc cgt aag att att att ggt 144Ala Gly Lys Val Leu Ala Arg His Gly Ser Arg Lys Ile Ile Ile Gly 35 40 45 aaa gac acg cgt att tct ggc tat atg ctg gag tca gca ctg gaa gcg 192Lys Asp Thr Arg Ile Ser Gly Tyr Met Leu Glu Ser Ala Leu Glu Ala 50 55 60 ggt ctg gcg gca gcg ggc ctt tcc gca ctc ttc act ggc ccg atg cca 240Gly Leu Ala Ala Ala Gly Leu Ser Ala Leu Phe Thr Gly Pro Met Pro 65 70 75 80 aca ccg gcc gtg gct tat ctg acg cgt acc ttc cgc gca gag gcc gga 288Thr Pro Ala Val Ala Tyr Leu Thr Arg Thr Phe Arg Ala Glu Ala Gly 85 90 95 att gtg ata tct gca tcg cat aac ccg ttc tac gat aat ggc att aaa 336Ile Val Ile Ser Ala Ser His Asn Pro Phe Tyr Asp Asn Gly Ile Lys 100 105 110 ttc ttc tct atc gac ggc acc aaa ctg ccg gat gcg gta gaa gag gcc 384Phe Phe Ser Ile Asp Gly Thr Lys Leu Pro Asp Ala Val Glu Glu Ala 115 120 125 atc gaa gcg gaa atg gaa aag gag atc agc tgc gtt gat tcg gca gaa 432Ile Glu Ala Glu Met Glu Lys Glu Ile Ser Cys Val Asp Ser Ala Glu 130 135 140 ctg ggt aaa gcc agc cgt atc gtt gat gcc gcg ggt cgc tat atc gag 480Leu Gly Lys Ala Ser Arg Ile Val Asp Ala Ala Gly Arg Tyr Ile Glu 145 150 155 160 ttt tgc aaa gcc acg ttc ccg aac gaa ctt agc ctc agt gaa ctg aag 528Phe Cys Lys Ala Thr Phe Pro Asn Glu Leu Ser Leu Ser Glu Leu Lys 165 170 175 att gtg gtg gat tgt gca aac ggt gcg act tat cac atc gcg ccg aac 576Ile Val Val Asp Cys Ala Asn Gly Ala Thr Tyr His Ile Ala Pro Asn 180 185 190 gtg ctg cgc gaa ctg ggg gcg aac gtt atc gct atc ggt tgt gag cca 624Val Leu Arg Glu Leu Gly Ala Asn Val Ile Ala Ile Gly Cys Glu Pro 195 200 205 aac ggt gta aac atc aat gcc gaa gtg ggg gct acc gac gtt cgc gcg 672Asn Gly Val Asn Ile Asn Ala Glu Val Gly Ala Thr Asp Val Arg Ala 210 215 220 ctc cag gct cgt gtg ctg gct gaa aaa gcg gat ctc ggt att gcc ttc 720Leu Gln Ala Arg Val Leu Ala Glu Lys Ala Asp Leu Gly Ile Ala Phe 225 230 235 240 gac ggc gat ggc gat cgc gtg att atg gtt gac cat gaa ggc aat aaa 768Asp Gly Asp Gly Asp Arg Val Ile Met Val Asp His Glu Gly Asn Lys 245 250 255 gtc gat ggc gat cag atc atg tat atc atc gcg cgt gaa ggt ctt cgt 816Val Asp Gly Asp Gln Ile Met Tyr Ile Ile Ala Arg Glu Gly Leu Arg 260 265 270 cag ggc cag ctg cgt ggt ggc gct gtg ggt aca ttg atg agc aac atg 864Gln Gly Gln Leu Arg Gly Gly Ala Val Gly Thr Leu Met Ser Asn Met 275 280 285 ggg ctt gaa ctg gcg ctg aaa cag tta gga att cca ttt gcg cgc gcg 912Gly Leu Glu Leu Ala Leu Lys Gln Leu Gly Ile Pro Phe Ala Arg Ala 290 295 300 aaa gtg ggt gac cgc tac gta ctg gaa aaa atg cag gag aaa ggc tgg 960Lys Val Gly Asp Arg Tyr Val Leu Glu Lys Met Gln Glu Lys Gly Trp 305 310 315 320 cgt atc ggt gca gag aat tcc ggt cat gtg atc ctg ctg gat aaa act 1008Arg Ile Gly Ala Glu Asn Ser Gly His Val Ile Leu Leu Asp Lys Thr 325 330 335 act acc ggt gac ggc atc gtt gct ggc ttg cag gtg ctg gcg gcg atg 1056Thr Thr Gly Asp Gly Ile Val Ala Gly Leu Gln Val Leu Ala Ala Met 340 345 350 gca cgt aac cat atg agc ctg cac gac ctt tgc agc ggc atg aaa atg 1104Ala Arg Asn His Met Ser Leu His Asp Leu Cys Ser Gly Met Lys Met 355 360 365 ttc ccg cag att ctg gtt aac gta cgt tac acc gca ggt agc ggc gat 1152Phe Pro Gln Ile Leu Val Asn Val Arg Tyr Thr Ala Gly Ser Gly Asp 370 375 380 cca ctt gag cat gag tca gtt aaa gcc gtg acc gca gag gtt gaa gct 1200Pro Leu Glu His Glu Ser Val Lys Ala Val Thr Ala Glu Val Glu Ala 385 390 395 400 gcg ctg ggc aac cgt gga cgc gtg ttg ctg cgt aaa tcc ggc acc gaa 1248Ala Leu Gly Asn Arg Gly Arg Val Leu Leu Arg Lys Ser Gly Thr Glu 405 410 415 ccg tta att cgc gtg atg gtg gaa ggc gaa gac gaa gcg cag gtg act 1296Pro Leu Ile Arg Val Met Val Glu Gly Glu Asp Glu Ala Gln Val Thr 420 425 430 gaa ttt gca cac cgc atc gcc gat gca gta aaa gcc gtt taa 1338Glu Phe Ala His Arg Ile Ala Asp Ala Val Lys Ala Val 435 440 445 80445PRTEscherichia coli 80Met Ser Asn Arg Lys Tyr Phe Gly Thr Asp Gly Ile Arg Gly Arg Val 1 5 10 15 Gly Asp Ala Pro Ile Thr Pro Asp Phe Val Leu Lys Leu Gly Trp Ala 20 25 30 Ala Gly Lys Val Leu Ala Arg His Gly Ser Arg Lys Ile Ile Ile Gly 35 40 45 Lys Asp Thr Arg Ile Ser Gly Tyr Met Leu Glu Ser Ala Leu Glu Ala 50 55 60 Gly Leu Ala Ala Ala Gly Leu Ser Ala Leu Phe Thr Gly Pro Met Pro 65 70 75 80 Thr Pro Ala Val Ala Tyr Leu Thr Arg Thr Phe Arg Ala Glu Ala Gly 85 90 95 Ile Val Ile Ser Ala Ser His Asn Pro Phe Tyr Asp Asn Gly Ile Lys 100 105 110 Phe Phe Ser Ile Asp Gly Thr Lys Leu Pro Asp Ala Val Glu Glu Ala 115 120 125 Ile Glu Ala Glu Met Glu Lys Glu Ile Ser Cys Val Asp Ser Ala Glu 130 135 140 Leu Gly Lys Ala Ser Arg Ile Val Asp Ala Ala Gly Arg Tyr Ile Glu 145 150 155 160 Phe Cys Lys Ala

Thr Phe Pro Asn Glu Leu Ser Leu Ser Glu Leu Lys 165 170 175 Ile Val Val Asp Cys Ala Asn Gly Ala Thr Tyr His Ile Ala Pro Asn 180 185 190 Val Leu Arg Glu Leu Gly Ala Asn Val Ile Ala Ile Gly Cys Glu Pro 195 200 205 Asn Gly Val Asn Ile Asn Ala Glu Val Gly Ala Thr Asp Val Arg Ala 210 215 220 Leu Gln Ala Arg Val Leu Ala Glu Lys Ala Asp Leu Gly Ile Ala Phe 225 230 235 240 Asp Gly Asp Gly Asp Arg Val Ile Met Val Asp His Glu Gly Asn Lys 245 250 255 Val Asp Gly Asp Gln Ile Met Tyr Ile Ile Ala Arg Glu Gly Leu Arg 260 265 270 Gln Gly Gln Leu Arg Gly Gly Ala Val Gly Thr Leu Met Ser Asn Met 275 280 285 Gly Leu Glu Leu Ala Leu Lys Gln Leu Gly Ile Pro Phe Ala Arg Ala 290 295 300 Lys Val Gly Asp Arg Tyr Val Leu Glu Lys Met Gln Glu Lys Gly Trp 305 310 315 320 Arg Ile Gly Ala Glu Asn Ser Gly His Val Ile Leu Leu Asp Lys Thr 325 330 335 Thr Thr Gly Asp Gly Ile Val Ala Gly Leu Gln Val Leu Ala Ala Met 340 345 350 Ala Arg Asn His Met Ser Leu His Asp Leu Cys Ser Gly Met Lys Met 355 360 365 Phe Pro Gln Ile Leu Val Asn Val Arg Tyr Thr Ala Gly Ser Gly Asp 370 375 380 Pro Leu Glu His Glu Ser Val Lys Ala Val Thr Ala Glu Val Glu Ala 385 390 395 400 Ala Leu Gly Asn Arg Gly Arg Val Leu Leu Arg Lys Ser Gly Thr Glu 405 410 415 Pro Leu Ile Arg Val Met Val Glu Gly Glu Asp Glu Ala Gln Val Thr 420 425 430 Glu Phe Ala His Arg Ile Ala Asp Ala Val Lys Ala Val 435 440 445 81858DNAEscherichia coliCDS(1)..(858)murI gene encoding a glutamase racemase 81atg gct acc aaa ctg cag gac ggg aat aca cct tgt ctg gca gct aca 48Met Ala Thr Lys Leu Gln Asp Gly Asn Thr Pro Cys Leu Ala Ala Thr 1 5 10 15 cct tct gaa cca cgt ccc acc gtg ctg gtg ttt gac tcc ggc gtc ggt 96Pro Ser Glu Pro Arg Pro Thr Val Leu Val Phe Asp Ser Gly Val Gly 20 25 30 ggg ttg tcg gtc tat gac gag atc cgg cat ctc tta ccg gat ctc cat 144Gly Leu Ser Val Tyr Asp Glu Ile Arg His Leu Leu Pro Asp Leu His 35 40 45 tac att tat gct ttc gat aac gtc gct ttc ccg tat ggc gaa aaa agc 192Tyr Ile Tyr Ala Phe Asp Asn Val Ala Phe Pro Tyr Gly Glu Lys Ser 50 55 60 gaa gcg ttt att gtt gag cga gtg gtg gca att gtc acc gcg gtg caa 240Glu Ala Phe Ile Val Glu Arg Val Val Ala Ile Val Thr Ala Val Gln 65 70 75 80 gaa cgt tat ccc ctt gcg ctg gct gtg gtc gct tgc aac act gcc agt 288Glu Arg Tyr Pro Leu Ala Leu Ala Val Val Ala Cys Asn Thr Ala Ser 85 90 95 acc gtt tca ctt cct gca tta cgc gaa aag ttc gac ttc ccg gtt gtt 336Thr Val Ser Leu Pro Ala Leu Arg Glu Lys Phe Asp Phe Pro Val Val 100 105 110 ggt gtc gtg ccg gcg att aaa cct gct gca cgt ctg acg gca aat ggc 384Gly Val Val Pro Ala Ile Lys Pro Ala Ala Arg Leu Thr Ala Asn Gly 115 120 125 att gtc gga tta ctg gca acc cgc gga aca gtt aaa cgt tct tat act 432Ile Val Gly Leu Leu Ala Thr Arg Gly Thr Val Lys Arg Ser Tyr Thr 130 135 140 cat gag ctg atc gcg cgt ttc gct aat gaa tgc cag ata gaa atg ctg 480His Glu Leu Ile Ala Arg Phe Ala Asn Glu Cys Gln Ile Glu Met Leu 145 150 155 160 ggc tcg gca gag atg gtt gag ttg gct gaa gcg aag cta cat ggc gaa 528Gly Ser Ala Glu Met Val Glu Leu Ala Glu Ala Lys Leu His Gly Glu 165 170 175 gat gtt tct ctg gat gca cta aaa cgt atc cta cgc ccg tgg tta aga 576Asp Val Ser Leu Asp Ala Leu Lys Arg Ile Leu Arg Pro Trp Leu Arg 180 185 190 atg aaa gag ccg cca gat acc gtt gta ttg ggt tgc acc cat ttc cct 624Met Lys Glu Pro Pro Asp Thr Val Val Leu Gly Cys Thr His Phe Pro 195 200 205 cta cta caa gaa gaa ctg tta caa gtg ctg cca gag gga acc cgg ctg 672Leu Leu Gln Glu Glu Leu Leu Gln Val Leu Pro Glu Gly Thr Arg Leu 210 215 220 gtg gat tct ggc gca gcg att gct cgc cga acg gcc tgg ttg tta gaa 720Val Asp Ser Gly Ala Ala Ile Ala Arg Arg Thr Ala Trp Leu Leu Glu 225 230 235 240 cat gaa gcc ccg gat gca aaa tct gcc gat gcg aat att gcc ttt tgt 768His Glu Ala Pro Asp Ala Lys Ser Ala Asp Ala Asn Ile Ala Phe Cys 245 250 255 atg gca atg acg cca gga gct gaa caa tta ttg ccc gtt tta cag cgt 816Met Ala Met Thr Pro Gly Ala Glu Gln Leu Leu Pro Val Leu Gln Arg 260 265 270 tac ggc ttc gaa acg ctc gaa aaa ctg gca gtt tta ggc tga 858Tyr Gly Phe Glu Thr Leu Glu Lys Leu Ala Val Leu Gly 275 280 285 82285PRTEscherichia coli 82Met Ala Thr Lys Leu Gln Asp Gly Asn Thr Pro Cys Leu Ala Ala Thr 1 5 10 15 Pro Ser Glu Pro Arg Pro Thr Val Leu Val Phe Asp Ser Gly Val Gly 20 25 30 Gly Leu Ser Val Tyr Asp Glu Ile Arg His Leu Leu Pro Asp Leu His 35 40 45 Tyr Ile Tyr Ala Phe Asp Asn Val Ala Phe Pro Tyr Gly Glu Lys Ser 50 55 60 Glu Ala Phe Ile Val Glu Arg Val Val Ala Ile Val Thr Ala Val Gln 65 70 75 80 Glu Arg Tyr Pro Leu Ala Leu Ala Val Val Ala Cys Asn Thr Ala Ser 85 90 95 Thr Val Ser Leu Pro Ala Leu Arg Glu Lys Phe Asp Phe Pro Val Val 100 105 110 Gly Val Val Pro Ala Ile Lys Pro Ala Ala Arg Leu Thr Ala Asn Gly 115 120 125 Ile Val Gly Leu Leu Ala Thr Arg Gly Thr Val Lys Arg Ser Tyr Thr 130 135 140 His Glu Leu Ile Ala Arg Phe Ala Asn Glu Cys Gln Ile Glu Met Leu 145 150 155 160 Gly Ser Ala Glu Met Val Glu Leu Ala Glu Ala Lys Leu His Gly Glu 165 170 175 Asp Val Ser Leu Asp Ala Leu Lys Arg Ile Leu Arg Pro Trp Leu Arg 180 185 190 Met Lys Glu Pro Pro Asp Thr Val Val Leu Gly Cys Thr His Phe Pro 195 200 205 Leu Leu Gln Glu Glu Leu Leu Gln Val Leu Pro Glu Gly Thr Arg Leu 210 215 220 Val Asp Ser Gly Ala Ala Ile Ala Arg Arg Thr Ala Trp Leu Leu Glu 225 230 235 240 His Glu Ala Pro Asp Ala Lys Ser Ala Asp Ala Asn Ile Ala Phe Cys 245 250 255 Met Ala Met Thr Pro Gly Ala Glu Gln Leu Leu Pro Val Leu Gln Arg 260 265 270 Tyr Gly Phe Glu Thr Leu Glu Lys Leu Ala Val Leu Gly 275 280 285 83591DNAEscherichia coliCDS(1)..(591)ribA gene encoding a GTP cyclohydrolase II 83atg cag ctt aaa cgt gtg gca gaa gcc aaa ctg cca acc cca tgg ggc 48Met Gln Leu Lys Arg Val Ala Glu Ala Lys Leu Pro Thr Pro Trp Gly 1 5 10 15 gat ttc ctg atg gtg gga ttt gaa gaa ctg gca acc gga cac gat cat 96Asp Phe Leu Met Val Gly Phe Glu Glu Leu Ala Thr Gly His Asp His 20 25 30 gtc gcg cta gtc tat ggc gat att tcc ggg cat acc ccg gta ctt gcg 144Val Ala Leu Val Tyr Gly Asp Ile Ser Gly His Thr Pro Val Leu Ala 35 40 45 cgc gtc cat tcc gaa tgt ctg acc ggt gac gcc ctg ttc agc ttg cgc 192Arg Val His Ser Glu Cys Leu Thr Gly Asp Ala Leu Phe Ser Leu Arg 50 55 60 tgc gat tgt ggc ttc cag ctc gaa gcg gca ttg acg caa att gcc gag 240Cys Asp Cys Gly Phe Gln Leu Glu Ala Ala Leu Thr Gln Ile Ala Glu 65 70 75 80 gaa ggc cgt ggt att ttg ctg tat cac cgt cag gaa ggt cgt aac att 288Glu Gly Arg Gly Ile Leu Leu Tyr His Arg Gln Glu Gly Arg Asn Ile 85 90 95 ggt ctg ctg aat aaa atc cgc gct tac gca ctg cag gat caa ggt tac 336Gly Leu Leu Asn Lys Ile Arg Ala Tyr Ala Leu Gln Asp Gln Gly Tyr 100 105 110 gat acc gta gag gct aac cac cag tta ggc ttc gcc gct gat gag cgc 384Asp Thr Val Glu Ala Asn His Gln Leu Gly Phe Ala Ala Asp Glu Arg 115 120 125 gac ttc act ctt tgc gct gat atg ttc aaa ctc ctt ggc gtc aat gaa 432Asp Phe Thr Leu Cys Ala Asp Met Phe Lys Leu Leu Gly Val Asn Glu 130 135 140 gtc cgc ttg tta acc aat aac ccg aaa aaa gtc gaa att ctg acc gaa 480Val Arg Leu Leu Thr Asn Asn Pro Lys Lys Val Glu Ile Leu Thr Glu 145 150 155 160 gca ggg att aat att gtt gaa cgc gta cca ttg att gta ggt cgt aac 528Ala Gly Ile Asn Ile Val Glu Arg Val Pro Leu Ile Val Gly Arg Asn 165 170 175 ccc aat aac gaa cat tat ctc gat acc aaa gcc gag aaa atg ggc cat 576Pro Asn Asn Glu His Tyr Leu Asp Thr Lys Ala Glu Lys Met Gly His 180 185 190 ttg ctg aac aaa taa 591Leu Leu Asn Lys 195 84196PRTEscherichia coli 84Met Gln Leu Lys Arg Val Ala Glu Ala Lys Leu Pro Thr Pro Trp Gly 1 5 10 15 Asp Phe Leu Met Val Gly Phe Glu Glu Leu Ala Thr Gly His Asp His 20 25 30 Val Ala Leu Val Tyr Gly Asp Ile Ser Gly His Thr Pro Val Leu Ala 35 40 45 Arg Val His Ser Glu Cys Leu Thr Gly Asp Ala Leu Phe Ser Leu Arg 50 55 60 Cys Asp Cys Gly Phe Gln Leu Glu Ala Ala Leu Thr Gln Ile Ala Glu 65 70 75 80 Glu Gly Arg Gly Ile Leu Leu Tyr His Arg Gln Glu Gly Arg Asn Ile 85 90 95 Gly Leu Leu Asn Lys Ile Arg Ala Tyr Ala Leu Gln Asp Gln Gly Tyr 100 105 110 Asp Thr Val Glu Ala Asn His Gln Leu Gly Phe Ala Ala Asp Glu Arg 115 120 125 Asp Phe Thr Leu Cys Ala Asp Met Phe Lys Leu Leu Gly Val Asn Glu 130 135 140 Val Arg Leu Leu Thr Asn Asn Pro Lys Lys Val Glu Ile Leu Thr Glu 145 150 155 160 Ala Gly Ile Asn Ile Val Glu Arg Val Pro Leu Ile Val Gly Arg Asn 165 170 175 Pro Asn Asn Glu His Tyr Leu Asp Thr Lys Ala Glu Lys Met Gly His 180 185 190 Leu Leu Asn Lys 195 85645DNAEscherichia coliCDS(1)..(645)adk gene encoding an adenylate kinase 85atg cgt atc att ctg ctt ggc gct ccg ggc gcg ggg aaa ggg act cag 48Met Arg Ile Ile Leu Leu Gly Ala Pro Gly Ala Gly Lys Gly Thr Gln 1 5 10 15 gct cag ttc atc atg gag aaa tat ggt att ccg caa atc tcc act ggc 96Ala Gln Phe Ile Met Glu Lys Tyr Gly Ile Pro Gln Ile Ser Thr Gly 20 25 30 gat atg ctg cgt gct gcg gtc aaa tct ggc tcc gag ctg ggt aaa caa 144Asp Met Leu Arg Ala Ala Val Lys Ser Gly Ser Glu Leu Gly Lys Gln 35 40 45 gca aaa gac att atg gat gct ggc aaa ctg gtc acc gac gaa ctg gtg 192Ala Lys Asp Ile Met Asp Ala Gly Lys Leu Val Thr Asp Glu Leu Val 50 55 60 atc gcg ctg gtt aaa gag cgc att gct cag gaa gac tgc cgt aat ggt 240Ile Ala Leu Val Lys Glu Arg Ile Ala Gln Glu Asp Cys Arg Asn Gly 65 70 75 80 ttc ctg ttg gac ggc ttc ccg cgt acc att ccg cag gca gac gcg atg 288Phe Leu Leu Asp Gly Phe Pro Arg Thr Ile Pro Gln Ala Asp Ala Met 85 90 95 aaa gaa gcg ggc atc aat gtt gat tac gtt ctg gaa ttc gac gta ccg 336Lys Glu Ala Gly Ile Asn Val Asp Tyr Val Leu Glu Phe Asp Val Pro 100 105 110 gac gaa ctg atc gtt gac cgt atc gtc ggt cgc cgc gtt cat gcg ccg 384Asp Glu Leu Ile Val Asp Arg Ile Val Gly Arg Arg Val His Ala Pro 115 120 125 tct ggt cgt gtt tat cac gtt aaa ttc aat ccg ccg aaa gta gaa ggc 432Ser Gly Arg Val Tyr His Val Lys Phe Asn Pro Pro Lys Val Glu Gly 130 135 140 aaa gac gac gtt acc ggt gaa gaa ctg act acc cgt aaa gat gat cag 480Lys Asp Asp Val Thr Gly Glu Glu Leu Thr Thr Arg Lys Asp Asp Gln 145 150 155 160 gaa gag acc gta cgt aaa cgt ctg gtt gaa tac cat cag atg aca gca 528Glu Glu Thr Val Arg Lys Arg Leu Val Glu Tyr His Gln Met Thr Ala 165 170 175 ccg ctg atc ggc tac tac tcc aaa gaa gca gaa gcg ggt aat acc aaa 576Pro Leu Ile Gly Tyr Tyr Ser Lys Glu Ala Glu Ala Gly Asn Thr Lys 180 185 190 tac gcg aaa gtt gac ggc acc aag ccg gtt gct gaa gtt cgc gct gat 624Tyr Ala Lys Val Asp Gly Thr Lys Pro Val Ala Glu Val Arg Ala Asp 195 200 205 ctg gaa aaa atc ctc ggc taa 645Leu Glu Lys Ile Leu Gly 210 86214PRTEscherichia coli 86Met Arg Ile Ile Leu Leu Gly Ala Pro Gly Ala Gly Lys Gly Thr Gln 1 5 10 15 Ala Gln Phe Ile Met Glu Lys Tyr Gly Ile Pro Gln Ile Ser Thr Gly 20 25 30 Asp Met Leu Arg Ala Ala Val Lys Ser Gly Ser Glu Leu Gly Lys Gln 35 40 45 Ala Lys Asp Ile Met Asp Ala Gly Lys Leu Val Thr Asp Glu Leu Val 50 55 60 Ile Ala Leu Val Lys Glu Arg Ile Ala Gln Glu Asp Cys Arg Asn Gly 65 70 75 80 Phe Leu Leu Asp Gly Phe Pro Arg Thr Ile Pro Gln Ala Asp Ala Met 85 90 95 Lys Glu Ala Gly Ile Asn Val Asp Tyr Val Leu Glu Phe Asp Val Pro 100 105 110 Asp Glu Leu Ile Val Asp Arg Ile Val Gly Arg Arg Val His Ala Pro 115 120 125 Ser Gly Arg Val Tyr His Val Lys Phe Asn Pro Pro Lys Val Glu Gly 130 135 140 Lys Asp Asp Val Thr Gly Glu Glu Leu Thr Thr Arg Lys Asp Asp Gln 145 150 155 160 Glu Glu Thr Val Arg Lys Arg Leu Val Glu Tyr His Gln Met Thr Ala 165 170 175 Pro Leu Ile Gly Tyr Tyr Ser Lys Glu Ala Glu Ala Gly Asn Thr Lys 180 185 190 Tyr Ala Lys Val Asp Gly Thr Lys Pro Val Ala Glu Val Arg Ala Asp 195 200 205 Leu Glu Lys Ile Leu Gly 210 871338DNAEscherichia coliCDS(1)..(1338)folp encodes dihydropteroate synthase 87atg agt aat cgt aaa tat ttc ggt acc gat ggg att cgt ggt cgt gta 48Met Ser Asn Arg Lys Tyr Phe Gly Thr Asp Gly Ile Arg Gly Arg Val 1 5 10 15 ggg gat gcg ccg atc aca cct gat ttt gtg ctt aag ctg ggt tgg gcc 96Gly Asp Ala Pro Ile Thr Pro Asp Phe Val Leu Lys Leu Gly Trp Ala 20

25 30 gcg ggt aaa gtg ctg gcg cgc cac ggc tcc cgt aag att att att ggt 144Ala Gly Lys Val Leu Ala Arg His Gly Ser Arg Lys Ile Ile Ile Gly 35 40 45 aaa gac acg cgt att tct ggc tat atg ctg gag tca gca ctg gaa gcg 192Lys Asp Thr Arg Ile Ser Gly Tyr Met Leu Glu Ser Ala Leu Glu Ala 50 55 60 ggt ctg gcg gca gcg ggc ctt tcc gca ctc ttc act ggc ccg atg cca 240Gly Leu Ala Ala Ala Gly Leu Ser Ala Leu Phe Thr Gly Pro Met Pro 65 70 75 80 aca ccg gcc gtg gct tat ctg acg cgt acc ttc cgc gca gag gcc gga 288Thr Pro Ala Val Ala Tyr Leu Thr Arg Thr Phe Arg Ala Glu Ala Gly 85 90 95 att gtg ata tct gca tcg cat aac ccg ttc tac gat aat ggc att aaa 336Ile Val Ile Ser Ala Ser His Asn Pro Phe Tyr Asp Asn Gly Ile Lys 100 105 110 ttc ttc tct atc gac ggc acc aaa ctg ccg gat gcg gta gaa gag gcc 384Phe Phe Ser Ile Asp Gly Thr Lys Leu Pro Asp Ala Val Glu Glu Ala 115 120 125 atc gaa gcg gaa atg gaa aag gag atc agc tgc gtt gat tcg gca gaa 432Ile Glu Ala Glu Met Glu Lys Glu Ile Ser Cys Val Asp Ser Ala Glu 130 135 140 ctg ggt aaa gcc agc cgt atc gtt gat gcc gcg ggt cgc tat atc gag 480Leu Gly Lys Ala Ser Arg Ile Val Asp Ala Ala Gly Arg Tyr Ile Glu 145 150 155 160 ttt tgc aaa gcc acg ttc ccg aac gaa ctt agc ctc agt gaa ctg aag 528Phe Cys Lys Ala Thr Phe Pro Asn Glu Leu Ser Leu Ser Glu Leu Lys 165 170 175 att gtg gtg gat tgt gca aac ggt gcg act tat cac atc gcg ccg aac 576Ile Val Val Asp Cys Ala Asn Gly Ala Thr Tyr His Ile Ala Pro Asn 180 185 190 gtg ctg cgc gaa ctg ggg gcg aac gtt atc gct atc ggt tgt gag cca 624Val Leu Arg Glu Leu Gly Ala Asn Val Ile Ala Ile Gly Cys Glu Pro 195 200 205 aac ggt gta aac atc aat gcc gaa gtg ggg gct acc gac gtt cgc gcg 672Asn Gly Val Asn Ile Asn Ala Glu Val Gly Ala Thr Asp Val Arg Ala 210 215 220 ctc cag gct cgt gtg ctg gct gaa aaa gcg gat ctc ggt att gcc ttc 720Leu Gln Ala Arg Val Leu Ala Glu Lys Ala Asp Leu Gly Ile Ala Phe 225 230 235 240 gac ggc gat ggc gat cgc gtg att atg gtt gac cat gaa ggc aat aaa 768Asp Gly Asp Gly Asp Arg Val Ile Met Val Asp His Glu Gly Asn Lys 245 250 255 gtc gat ggc gat cag atc atg tat atc atc gcg cgt gaa ggt ctt cgt 816Val Asp Gly Asp Gln Ile Met Tyr Ile Ile Ala Arg Glu Gly Leu Arg 260 265 270 cag ggc cag ctg cgt ggt ggc gct gtg ggt aca ttg atg agc aac atg 864Gln Gly Gln Leu Arg Gly Gly Ala Val Gly Thr Leu Met Ser Asn Met 275 280 285 ggg ctt gaa ctg gcg ctg aaa cag tta gga att cca ttt gcg cgc gcg 912Gly Leu Glu Leu Ala Leu Lys Gln Leu Gly Ile Pro Phe Ala Arg Ala 290 295 300 aaa gtg ggt gac cgc tac gta ctg gaa aaa atg cag gag aaa ggc tgg 960Lys Val Gly Asp Arg Tyr Val Leu Glu Lys Met Gln Glu Lys Gly Trp 305 310 315 320 cgt atc ggt gca gag aat tcc ggt cat gtg atc ctg ctg gat aaa act 1008Arg Ile Gly Ala Glu Asn Ser Gly His Val Ile Leu Leu Asp Lys Thr 325 330 335 act acc ggt gac ggc atc gtt gct ggc ttg cag gtg ctg gcg gcg atg 1056Thr Thr Gly Asp Gly Ile Val Ala Gly Leu Gln Val Leu Ala Ala Met 340 345 350 gca cgt aac cat atg agc ctg cac gac ctt tgc agc ggc atg aaa atg 1104Ala Arg Asn His Met Ser Leu His Asp Leu Cys Ser Gly Met Lys Met 355 360 365 ttc ccg cag att ctg gtt aac gta cgt tac acc gca ggt agc ggc gat 1152Phe Pro Gln Ile Leu Val Asn Val Arg Tyr Thr Ala Gly Ser Gly Asp 370 375 380 cca ctt gag cat gag tca gtt aaa gcc gtg acc gca gag gtt gaa gct 1200Pro Leu Glu His Glu Ser Val Lys Ala Val Thr Ala Glu Val Glu Ala 385 390 395 400 gcg ctg ggc aac cgt gga cgc gtg ttg ctg cgt aaa tcc ggc acc gaa 1248Ala Leu Gly Asn Arg Gly Arg Val Leu Leu Arg Lys Ser Gly Thr Glu 405 410 415 ccg tta att cgc gtg atg gtg gaa ggc gaa gac gaa gcg cag gtg act 1296Pro Leu Ile Arg Val Met Val Glu Gly Glu Asp Glu Ala Gln Val Thr 420 425 430 gaa ttt gca cac cgc atc gcc gat gca gta aaa gcc gtt taa 1338Glu Phe Ala His Arg Ile Ala Asp Ala Val Lys Ala Val 435 440 445 88445PRTEscherichia coli 88Met Ser Asn Arg Lys Tyr Phe Gly Thr Asp Gly Ile Arg Gly Arg Val 1 5 10 15 Gly Asp Ala Pro Ile Thr Pro Asp Phe Val Leu Lys Leu Gly Trp Ala 20 25 30 Ala Gly Lys Val Leu Ala Arg His Gly Ser Arg Lys Ile Ile Ile Gly 35 40 45 Lys Asp Thr Arg Ile Ser Gly Tyr Met Leu Glu Ser Ala Leu Glu Ala 50 55 60 Gly Leu Ala Ala Ala Gly Leu Ser Ala Leu Phe Thr Gly Pro Met Pro 65 70 75 80 Thr Pro Ala Val Ala Tyr Leu Thr Arg Thr Phe Arg Ala Glu Ala Gly 85 90 95 Ile Val Ile Ser Ala Ser His Asn Pro Phe Tyr Asp Asn Gly Ile Lys 100 105 110 Phe Phe Ser Ile Asp Gly Thr Lys Leu Pro Asp Ala Val Glu Glu Ala 115 120 125 Ile Glu Ala Glu Met Glu Lys Glu Ile Ser Cys Val Asp Ser Ala Glu 130 135 140 Leu Gly Lys Ala Ser Arg Ile Val Asp Ala Ala Gly Arg Tyr Ile Glu 145 150 155 160 Phe Cys Lys Ala Thr Phe Pro Asn Glu Leu Ser Leu Ser Glu Leu Lys 165 170 175 Ile Val Val Asp Cys Ala Asn Gly Ala Thr Tyr His Ile Ala Pro Asn 180 185 190 Val Leu Arg Glu Leu Gly Ala Asn Val Ile Ala Ile Gly Cys Glu Pro 195 200 205 Asn Gly Val Asn Ile Asn Ala Glu Val Gly Ala Thr Asp Val Arg Ala 210 215 220 Leu Gln Ala Arg Val Leu Ala Glu Lys Ala Asp Leu Gly Ile Ala Phe 225 230 235 240 Asp Gly Asp Gly Asp Arg Val Ile Met Val Asp His Glu Gly Asn Lys 245 250 255 Val Asp Gly Asp Gln Ile Met Tyr Ile Ile Ala Arg Glu Gly Leu Arg 260 265 270 Gln Gly Gln Leu Arg Gly Gly Ala Val Gly Thr Leu Met Ser Asn Met 275 280 285 Gly Leu Glu Leu Ala Leu Lys Gln Leu Gly Ile Pro Phe Ala Arg Ala 290 295 300 Lys Val Gly Asp Arg Tyr Val Leu Glu Lys Met Gln Glu Lys Gly Trp 305 310 315 320 Arg Ile Gly Ala Glu Asn Ser Gly His Val Ile Leu Leu Asp Lys Thr 325 330 335 Thr Thr Gly Asp Gly Ile Val Ala Gly Leu Gln Val Leu Ala Ala Met 340 345 350 Ala Arg Asn His Met Ser Leu His Asp Leu Cys Ser Gly Met Lys Met 355 360 365 Phe Pro Gln Ile Leu Val Asn Val Arg Tyr Thr Ala Gly Ser Gly Asp 370 375 380 Pro Leu Glu His Glu Ser Val Lys Ala Val Thr Ala Glu Val Glu Ala 385 390 395 400 Ala Leu Gly Asn Arg Gly Arg Val Leu Leu Arg Lys Ser Gly Thr Glu 405 410 415 Pro Leu Ile Arg Val Met Val Glu Gly Glu Asp Glu Ala Gln Val Thr 420 425 430 Glu Phe Ala His Arg Ile Ala Asp Ala Val Lys Ala Val 435 440 445 8935DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideprimer_bind(1)..(35)J23118 primer 89ttgacggcta gctcagtcct aggtattgtg ctagc 3590142DNAEscherichia colimisc_feature(1)..(142)thiC gene region corresponding to 5'UTRmutation(8)..(8)Mutation; wherein B=T or C or G 90attcgggbtc cgcggaacct gatcaggcta atacctgcga agggaacaag agttaatctg 60ctatcgcatc gcccctgcgg cgatcgtctc ttgcttcatc cgtcgtctga caagccacgt 120ccttaacttt ttggaatgag ct 142912283DNAEscherichia colipromoter(1665)..(1990)pBAD promoterRBS(1980)..(1985)CDS(1991)..(2281)Portion of folP gene sequence 91aagatgctct catgaaatat gagactatcg acgcaccgca gattgatgac ctgatggcac 60gtcgcgatgt acgtccgcca gcgggctggg aagaaccagg cgcttctaac aattctggcg 120acaatggtag tccaaaggct cctcgtccgg ttgatgaacc gcgtacgccg aacccgggta 180acaccatgtc agagcagtta ggcgacaagt aagttcccgc atcagatgac tgtatttgta 240ccgaaaaccc cggggcgtgc tccggggttt tttcttatca attcatacca gggataacat 300cagcgattgt gtaggctgga gctgcttcga agttcctata ctttctagag aataggaact 360tcggaatagg aacttcaaga tccccttatt agaagaactc gtcaagaagg cgatagaagg 420cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt 480cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg 540ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat 600tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct 660tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct 720gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt 780ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga 840tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc 900ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa 960cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac 1020cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg 1080cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc 1140aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc 1200ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca 1260tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg 1320gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag 1380ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat 1440tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc 1500agcccttgcg ccctgagtgc ttgcggcagc gtgagcttca aaagcgctct gaagttccta 1560tactttctag agaataggaa cttcgaactg caggtcgacg gatccccgga atattcatat 1620gtgcgcttca gccatacttt tcatactccc gccattcaga gaagaaacca attgtccata 1680ttgcatcaga cattgccgtc actgcgtctt ttactggctc ttctcgctaa ccaaaccggt 1740aaccccgctt attaaaagca ttctgtaaca aagcgggacc aaagccatga caaaaacgcg 1800taacaaaagt gtctataatc acggcagaaa agtccacatt gattatttgc acggcgtcac 1860actttgctat gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt 1920atcgcaactc tctactgttt ctccataccc gtttttttgg gctagcgaat tcgagctcga 1980cttgcaaggt atg aaa ctc ttt gcc cag ggt act tca ctg gac ctt agc 2029 Met Lys Leu Phe Ala Gln Gly Thr Ser Leu Asp Leu Ser 1 5 10 cat cct cac gta atg ggg atc ctc aac gtc acg cct gat tcc ttt tcg 2077His Pro His Val Met Gly Ile Leu Asn Val Thr Pro Asp Ser Phe Ser 15 20 25 gat ggt ggc acg cat aac tcg ctg ata gat gcg gtg aaa cat gcg aat 2125Asp Gly Gly Thr His Asn Ser Leu Ile Asp Ala Val Lys His Ala Asn 30 35 40 45 ctg atg atc aac gct ggc gcg acg atc att gac gtt ggt ggc gag tcc 2173Leu Met Ile Asn Ala Gly Ala Thr Ile Ile Asp Val Gly Gly Glu Ser 50 55 60 acg cgc cca ggg gcg gcg gaa gtt agc gtt gaa gaa gag ttg caa cgt 2221Thr Arg Pro Gly Ala Ala Glu Val Ser Val Glu Glu Glu Leu Gln Arg 65 70 75 gtt att cct gtg gtt gag gca att gct caa cgc ttc gaa gtc tgg atc 2269Val Ile Pro Val Val Glu Ala Ile Ala Gln Arg Phe Glu Val Trp Ile 80 85 90 tca gtc gat aca tc 2283Ser Val Asp Thr 95 9297PRTEscherichia coli 92Met Lys Leu Phe Ala Gln Gly Thr Ser Leu Asp Leu Ser His Pro His 1 5 10 15 Val Met Gly Ile Leu Asn Val Thr Pro Asp Ser Phe Ser Asp Gly Gly 20 25 30 Thr His Asn Ser Leu Ile Asp Ala Val Lys His Ala Asn Leu Met Ile 35 40 45 Asn Ala Gly Ala Thr Ile Ile Asp Val Gly Gly Glu Ser Thr Arg Pro 50 55 60 Gly Ala Ala Glu Val Ser Val Glu Glu Glu Leu Gln Arg Val Ile Pro 65 70 75 80 Val Val Glu Ala Ile Ala Gln Arg Phe Glu Val Trp Ile Ser Val Asp 85 90 95 Thr 934790DNAEscherichia coliCDS(1945)..(2916)SpecR gene in pBALspec. 93atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660tgccagtagg cgcgcggacg aaagtaaacc cactggcgat accattcgcg agcctccgga 720tgacgaccta agtgaccaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900gggattaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gacatcattt 960tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020tgcatacttt tcatactccc gccattcaga gaagaaacca attgtccata ttgcatcaga 1080cattgccgtc actgcgtctt ttactggctc ttctcgctaa ccaaaccggt aaccccgctt 1140attaaaagca ttctgtaaca aagcgggacc aaagccatga caaaaacgcg taacaaaagt 1200gtctataatc acggcagaaa agtccacatt gattatttgc acggcgtcac actttgctat 1260gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc 1320tctactgttt ctccataccc gtttttttgg gctagcgaat tcgagctcga ggaggaaacc 1380atggtacccg gggatcctct agagtcgacc tgcaggcatg caagcttggc tgttttggcg 1440gatgagagaa gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa 1500aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag 1560aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact 1620gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 1680tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt 1740gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa 1800attaagcaga aggccatcct gacggtgata tccggatgaa ggcacgaacc cagtggacat 1860aagcctcgtt cggttcgtaa gctgtaatgc aagtagcgta actgccgtca cgcaactggt 1920ccacaacctt gaccgaacgc agcg gtg gta acg gcg cag tgg cgg ttt tca 1971 Val Val Thr Ala Gln Trp Arg Phe Ser 1 5 tgg ctt ctt gtt atg aca tgt ttt ttt ggg gta cag tct atg cct cgg 2019Trp Leu Leu Val Met Thr Cys Phe Phe Gly Val Gln Ser Met Pro Arg 10 15 20 25 gca tcc aag cag caa gcg cgt tac gcc gtg ggt cga tgt ttg atg tta 2067Ala Ser Lys Gln Gln Ala Arg Tyr Ala Val Gly Arg Cys Leu Met Leu 30 35 40 tgg agc agc aac gat gtt acg cag cag ggc agt cgc cct aaa aca aag 2115Trp Ser Ser Asn Asp Val Thr Gln Gln Gly Ser Arg Pro Lys Thr Lys 45 50 55 tta aac atc atg agg gaa gcg gtg atc gcc gaa gta tcg act caa cta 2163Leu Asn Ile Met Arg Glu Ala Val Ile Ala Glu Val Ser Thr Gln Leu 60 65 70 tca gag gta gtt ggc gtc atc gag cgc cat ctc gaa ccg acg ttg ctg

2211Ser Glu Val Val Gly Val Ile Glu Arg His Leu Glu Pro Thr Leu Leu 75 80 85 gcc gta cat ttg tac ggc tcc gca gtg gat ggc ggc ctg aag cca cac 2259Ala Val His Leu Tyr Gly Ser Ala Val Asp Gly Gly Leu Lys Pro His 90 95 100 105 agt gat att gat ttg ctg gtt acg gtg acc gta agg ctt gat gaa aca 2307Ser Asp Ile Asp Leu Leu Val Thr Val Thr Val Arg Leu Asp Glu Thr 110 115 120 acg cgg cga gct ttg atc aac gac ctt ttg gaa act tcg gct tcc cct 2355Thr Arg Arg Ala Leu Ile Asn Asp Leu Leu Glu Thr Ser Ala Ser Pro 125 130 135 gga gag agc gag att ctc cgc gct gta gaa gtc acc att gtt gtg cac 2403Gly Glu Ser Glu Ile Leu Arg Ala Val Glu Val Thr Ile Val Val His 140 145 150 gac gac atc att ccg tgg cgt tat cca gct aag cgc gaa ctg caa ttt 2451Asp Asp Ile Ile Pro Trp Arg Tyr Pro Ala Lys Arg Glu Leu Gln Phe 155 160 165 gga gaa tgg cag cgc aat gac att ctt gca ggt atc ttc gag cca gcc 2499Gly Glu Trp Gln Arg Asn Asp Ile Leu Ala Gly Ile Phe Glu Pro Ala 170 175 180 185 acg tac gac att gat ctg gct atc ttg ctg aca aaa gca aga gaa cat 2547Thr Tyr Asp Ile Asp Leu Ala Ile Leu Leu Thr Lys Ala Arg Glu His 190 195 200 agc gtt gcc ttg gta ggt cca gcg gcg gag gaa ctc ttt gat ccg gtt 2595Ser Val Ala Leu Val Gly Pro Ala Ala Glu Glu Leu Phe Asp Pro Val 205 210 215 cct gaa cag gat cta ttt gag gcg cta aat gaa acc tta acg cta tgg 2643Pro Glu Gln Asp Leu Phe Glu Ala Leu Asn Glu Thr Leu Thr Leu Trp 220 225 230 aac tcg ccg ccc gac tgg gct ggc gat gag cga aat gta gtg ctt acg 2691Asn Ser Pro Pro Asp Trp Ala Gly Asp Glu Arg Asn Val Val Leu Thr 235 240 245 ttg tcc cgc att tgg tac agc gca gta acc ggc aaa atc gcg ccg aag 2739Leu Ser Arg Ile Trp Tyr Ser Ala Val Thr Gly Lys Ile Ala Pro Lys 250 255 260 265 gat gtc gct gcc gac tgg gca atg gag cgc ctg ccg gcc cag tat cag 2787Asp Val Ala Ala Asp Trp Ala Met Glu Arg Leu Pro Ala Gln Tyr Gln 270 275 280 ccc gtc ata ctt gaa gct aga cag gct tat ctt gga caa gaa gaa gat 2835Pro Val Ile Leu Glu Ala Arg Gln Ala Tyr Leu Gly Gln Glu Glu Asp 285 290 295 cgc ttg gcc tcg cgc gca gat cag ttg gaa gaa ttt gtc cac tac gtg 2883Arg Leu Ala Ser Arg Ala Asp Gln Leu Glu Glu Phe Val His Tyr Val 300 305 310 aaa ggc gag atc acc aag gta gtc ggc aaa taa tgtctaacaa ttcgttcaag 2936Lys Gly Glu Ile Thr Lys Val Val Gly Lys 315 320 ccgacggata tctagattga tttacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 2996ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 3056tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 3116gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt 3176gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 3236ggagtccacg ttctttaata gtggactctt gttccaaact tgaacaacac tcaaccctat 3296ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 3356tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaattta 3416aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 3476tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 3536tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 3596gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 3656agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 3716tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 3776ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 3836cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 3896tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 3956acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 4016gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 4076ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 4136tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 4196attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 4256cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc 4316tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 4376ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc 4436tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 4496atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 4556gtcatcaccg aaacgcgcga ggcagcaagg agatggcgcc caacagtccc ccggccacgg 4616ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat 4676cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga 4736tgccggccac gatgcgtccg gcgtagagga tctgctcatg tttgacagct tatc 479094323PRTEscherichia coli 94Val Val Thr Ala Gln Trp Arg Phe Ser Trp Leu Leu Val Met Thr Cys 1 5 10 15 Phe Phe Gly Val Gln Ser Met Pro Arg Ala Ser Lys Gln Gln Ala Arg 20 25 30 Tyr Ala Val Gly Arg Cys Leu Met Leu Trp Ser Ser Asn Asp Val Thr 35 40 45 Gln Gln Gly Ser Arg Pro Lys Thr Lys Leu Asn Ile Met Arg Glu Ala 50 55 60 Val Ile Ala Glu Val Ser Thr Gln Leu Ser Glu Val Val Gly Val Ile 65 70 75 80 Glu Arg His Leu Glu Pro Thr Leu Leu Ala Val His Leu Tyr Gly Ser 85 90 95 Ala Val Asp Gly Gly Leu Lys Pro His Ser Asp Ile Asp Leu Leu Val 100 105 110 Thr Val Thr Val Arg Leu Asp Glu Thr Thr Arg Arg Ala Leu Ile Asn 115 120 125 Asp Leu Leu Glu Thr Ser Ala Ser Pro Gly Glu Ser Glu Ile Leu Arg 130 135 140 Ala Val Glu Val Thr Ile Val Val His Asp Asp Ile Ile Pro Trp Arg 145 150 155 160 Tyr Pro Ala Lys Arg Glu Leu Gln Phe Gly Glu Trp Gln Arg Asn Asp 165 170 175 Ile Leu Ala Gly Ile Phe Glu Pro Ala Thr Tyr Asp Ile Asp Leu Ala 180 185 190 Ile Leu Leu Thr Lys Ala Arg Glu His Ser Val Ala Leu Val Gly Pro 195 200 205 Ala Ala Glu Glu Leu Phe Asp Pro Val Pro Glu Gln Asp Leu Phe Glu 210 215 220 Ala Leu Asn Glu Thr Leu Thr Leu Trp Asn Ser Pro Pro Asp Trp Ala 225 230 235 240 Gly Asp Glu Arg Asn Val Val Leu Thr Leu Ser Arg Ile Trp Tyr Ser 245 250 255 Ala Val Thr Gly Lys Ile Ala Pro Lys Asp Val Ala Ala Asp Trp Ala 260 265 270 Met Glu Arg Leu Pro Ala Gln Tyr Gln Pro Val Ile Leu Glu Ala Arg 275 280 285 Gln Ala Tyr Leu Gly Gln Glu Glu Asp Arg Leu Ala Ser Arg Ala Asp 290 295 300 Gln Leu Glu Glu Phe Val His Tyr Val Lys Gly Glu Ile Thr Lys Val 305 310 315 320 Val Gly Lys 95930DNAEscherichia coliCDS(1)..(930)AraCtal gene encoding AraCtal biosensor 95atg caa tat gga caa ttg gtt tct tct ctg aat ggc ggg agt atg aaa 48Met Gln Tyr Gly Gln Leu Val Ser Ser Leu Asn Gly Gly Ser Met Lys 1 5 10 15 agt atg gct gaa gcg caa aat gat gtc ctg ctg ccg gga tac tcg ttt 96Ser Met Ala Glu Ala Gln Asn Asp Val Leu Leu Pro Gly Tyr Ser Phe 20 25 30 aat gcc cat ctg gtg gcg ggt tta atc ccg att gag gcc aac ggt tat 144Asn Ala His Leu Val Ala Gly Leu Ile Pro Ile Glu Ala Asn Gly Tyr 35 40 45 ctc gat ttt ttt atc gac cga ccg ctg gga atg aaa ggt tat att ctc 192Leu Asp Phe Phe Ile Asp Arg Pro Leu Gly Met Lys Gly Tyr Ile Leu 50 55 60 aat ctc acc att cgc ggt cag ggg gtg gtg aaa aat cag gga cga gaa 240Asn Leu Thr Ile Arg Gly Gln Gly Val Val Lys Asn Gln Gly Arg Glu 65 70 75 80 ttt gtt tgc cga ccg ggt gat att ttg ctg ttc ccg cca gga gag att 288Phe Val Cys Arg Pro Gly Asp Ile Leu Leu Phe Pro Pro Gly Glu Ile 85 90 95 ggt cac tta ggt cgt cat ccg gag gct cgc gaa tgg tat cgc cag tgg 336Gly His Leu Gly Arg His Pro Glu Ala Arg Glu Trp Tyr Arg Gln Trp 100 105 110 gtt tac ttt cgt ccg cgc gcc tac tgg cat gaa tgg ctt aac tgg ccg 384Val Tyr Phe Arg Pro Arg Ala Tyr Trp His Glu Trp Leu Asn Trp Pro 115 120 125 tca ata ttt gcc aat acg ggg ttc ttt cgc ccg gat gaa gcg cac cag 432Ser Ile Phe Ala Asn Thr Gly Phe Phe Arg Pro Asp Glu Ala His Gln 130 135 140 ccg cat ttc agc gac ctg ttt ggg caa atc att aac gcc ggg caa ggg 480Pro His Phe Ser Asp Leu Phe Gly Gln Ile Ile Asn Ala Gly Gln Gly 145 150 155 160 gaa ggg cgc tat tcg gag ctg ctg gcg ata aat ctg ctt gag caa ttg 528Glu Gly Arg Tyr Ser Glu Leu Leu Ala Ile Asn Leu Leu Glu Gln Leu 165 170 175 tta ctg cgg cgc atg gaa gcg att aac gag tcg ctc cat cca ccg atg 576Leu Leu Arg Arg Met Glu Ala Ile Asn Glu Ser Leu His Pro Pro Met 180 185 190 gat aat cgg gta cgc gag gct tgt cag tac atc agc gat cac ctg gca 624Asp Asn Arg Val Arg Glu Ala Cys Gln Tyr Ile Ser Asp His Leu Ala 195 200 205 gac agc aat ttt gat atc gcc agc gtc gca cag cat gtt tgc ttg tcg 672Asp Ser Asn Phe Asp Ile Ala Ser Val Ala Gln His Val Cys Leu Ser 210 215 220 ccg tcg cgt ctg tca cat ctt ttc cgc cag cag tta ggg att agc gtc 720Pro Ser Arg Leu Ser His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val 225 230 235 240 tta agc tgg cgc gag gac caa cgt atc agc cag gcg aag ctg ctt ttg 768Leu Ser Trp Arg Glu Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu 245 250 255 agc acc acc cgg atg cct atc gcc acc gtc ggt cgc aat gtt ggt ttt 816Ser Thr Thr Arg Met Pro Ile Ala Thr Val Gly Arg Asn Val Gly Phe 260 265 270 gac gat caa ctc tat ttc tcg cgg gta ttt aaa aaa tgc acc ggg gcc 864Asp Asp Gln Leu Tyr Phe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala 275 280 285 agc ccg agc gag ttc cgt gcc ggt tgt gaa gaa aaa gtg aat gat gta 912Ser Pro Ser Glu Phe Arg Ala Gly Cys Glu Glu Lys Val Asn Asp Val 290 295 300 gcc gtc aag ttg tca taa 930Ala Val Lys Leu Ser 305 96309PRTEscherichia coli 96Met Gln Tyr Gly Gln Leu Val Ser Ser Leu Asn Gly Gly Ser Met Lys 1 5 10 15 Ser Met Ala Glu Ala Gln Asn Asp Val Leu Leu Pro Gly Tyr Ser Phe 20 25 30 Asn Ala His Leu Val Ala Gly Leu Ile Pro Ile Glu Ala Asn Gly Tyr 35 40 45 Leu Asp Phe Phe Ile Asp Arg Pro Leu Gly Met Lys Gly Tyr Ile Leu 50 55 60 Asn Leu Thr Ile Arg Gly Gln Gly Val Val Lys Asn Gln Gly Arg Glu 65 70 75 80 Phe Val Cys Arg Pro Gly Asp Ile Leu Leu Phe Pro Pro Gly Glu Ile 85 90 95 Gly His Leu Gly Arg His Pro Glu Ala Arg Glu Trp Tyr Arg Gln Trp 100 105 110 Val Tyr Phe Arg Pro Arg Ala Tyr Trp His Glu Trp Leu Asn Trp Pro 115 120 125 Ser Ile Phe Ala Asn Thr Gly Phe Phe Arg Pro Asp Glu Ala His Gln 130 135 140 Pro His Phe Ser Asp Leu Phe Gly Gln Ile Ile Asn Ala Gly Gln Gly 145 150 155 160 Glu Gly Arg Tyr Ser Glu Leu Leu Ala Ile Asn Leu Leu Glu Gln Leu 165 170 175 Leu Leu Arg Arg Met Glu Ala Ile Asn Glu Ser Leu His Pro Pro Met 180 185 190 Asp Asn Arg Val Arg Glu Ala Cys Gln Tyr Ile Ser Asp His Leu Ala 195 200 205 Asp Ser Asn Phe Asp Ile Ala Ser Val Ala Gln His Val Cys Leu Ser 210 215 220 Pro Ser Arg Leu Ser His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val 225 230 235 240 Leu Ser Trp Arg Glu Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu 245 250 255 Ser Thr Thr Arg Met Pro Ile Ala Thr Val Gly Arg Asn Val Gly Phe 260 265 270 Asp Asp Gln Leu Tyr Phe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala 275 280 285 Ser Pro Ser Glu Phe Arg Ala Gly Cys Glu Glu Lys Val Asn Asp Val 290 295 300 Ala Val Lys Leu Ser 305 974452DNAEscherichia coliCDS(1607)..(2578)SpecR gene in pBANspec 97atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accactcaat ttcaatccgt aaacaggtca 120aacatcagtt gccgcaacca aatattggct aggtccttgt ggtacttcgc atgccagaac 180atgttgatgg ctatttcagg caagacgact gggtgcggca aggcgcttag gccgaagggc 240tccacgcagc agtcggctaa acgtatcggc acagtggcga gcagatcggt gcgctggagg 300atgtggccaa cggcggcgaa gtgcggcact tccagacgga tgtcgcgccg gatgccgacc 360cgtgtcatgt acgtgtccac ctcgccgtgg ccggtgccag cggcgatgac acgcacgtgg 420ccgtaggaac agaagcgctc cagagtcagg ggttcgcggg tgactggatg gtccttgcga 480cataggcaca cgtagtgatt ctggagcagc cggcgctgaa agaagccagt ttgcagattg 540ggaagcaggc ccacggccaa gtccacggtt ccgttctgca aggcctgcat caggctcatc 600gaactgtcgc gcaccgtact gatcacgcaa ttgggggcct ggtgagccag cacatccatc 660agccgcggca tgaagtagat ctcgccaatg tcggtcatgg ccagggtgaa ggtacgctcg 720ctggtcagcg gatcgaagct ttcatggtgc tgtagggcgt tgcgcagtgc gtgcatggcc 780gaagtgacgg gctcggccag atgcgcggca tagggtgtgg gttccattcc ctgatgtgtg 840cgcacgaaga gtgggtcctg tagcgaggtg cgcaggcgtt tcagcgcatt gctcacggca 900ggctgggtca ggcccaggtt ctccgcagtg atagagacgc gtctgtcgac cagcaactgg 960ttgaacacca ccagcaggtt taaatccagg tcacgcagtt ccatggggcc tcgcttgggt 1020tgctagcatt gtacctagga ctgagctagc cgtaaatcct ctagagtcga cctgcaggca 1080tgcaagcttg gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca 1140gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca 1200cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct 1260ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga 1320ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc 1380gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc 1440gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggtga tatccggatg 1500aaggcacgaa cccagtggac ataagcctcg ttcggttcgt aagctgtaat gcaagtagcg 1560taactgccgt cacgcaactg gtccacaacc ttgaccgaac gcagcg gtg gta acg 1615 Val Val Thr 1 gcg cag tgg cgg ttt tca tgg ctt ctt gtt atg aca tgt ttt ttt ggg 1663Ala Gln Trp Arg Phe Ser Trp Leu Leu Val Met Thr Cys Phe Phe Gly 5 10 15 gta cag tct atg cct cgg gca tcc aag cag caa gcg cgt tac gcc gtg 1711Val Gln Ser Met Pro Arg Ala Ser Lys Gln Gln Ala Arg Tyr Ala Val 20 25 30 35 ggt cga tgt ttg atg tta tgg agc agc aac gat gtt acg cag cag ggc 1759Gly Arg Cys Leu Met Leu Trp Ser Ser Asn Asp Val Thr Gln Gln Gly 40 45 50 agt cgc cct aaa aca aag tta aac atc atg agg gaa gcg gtg atc gcc 1807Ser Arg Pro Lys Thr Lys Leu Asn Ile Met Arg Glu Ala Val Ile Ala 55 60 65 gaa gta tcg act caa cta tca gag gta gtt ggc gtc atc gag cgc cat

1855Glu Val Ser Thr Gln Leu Ser Glu Val Val Gly Val Ile Glu Arg His 70 75 80 ctc gaa ccg acg ttg ctg gcc gta cat ttg tac ggc tcc gca gtg gat 1903Leu Glu Pro Thr Leu Leu Ala Val His Leu Tyr Gly Ser Ala Val Asp 85 90 95 ggc ggc ctg aag cca cac agt gat att gat ttg ctg gtt acg gtg acc 1951Gly Gly Leu Lys Pro His Ser Asp Ile Asp Leu Leu Val Thr Val Thr 100 105 110 115 gta agg ctt gat gaa aca acg cgg cga gct ttg atc aac gac ctt ttg 1999Val Arg Leu Asp Glu Thr Thr Arg Arg Ala Leu Ile Asn Asp Leu Leu 120 125 130 gaa act tcg gct tcc cct gga gag agc gag att ctc cgc gct gta gaa 2047Glu Thr Ser Ala Ser Pro Gly Glu Ser Glu Ile Leu Arg Ala Val Glu 135 140 145 gtc acc att gtt gtg cac gac gac atc att ccg tgg cgt tat cca gct 2095Val Thr Ile Val Val His Asp Asp Ile Ile Pro Trp Arg Tyr Pro Ala 150 155 160 aag cgc gaa ctg caa ttt gga gaa tgg cag cgc aat gac att ctt gca 2143Lys Arg Glu Leu Gln Phe Gly Glu Trp Gln Arg Asn Asp Ile Leu Ala 165 170 175 ggt atc ttc gag cca gcc acg tac gac att gat ctg gct atc ttg ctg 2191Gly Ile Phe Glu Pro Ala Thr Tyr Asp Ile Asp Leu Ala Ile Leu Leu 180 185 190 195 aca aaa gca aga gaa cat agc gtt gcc ttg gta ggt cca gcg gcg gag 2239Thr Lys Ala Arg Glu His Ser Val Ala Leu Val Gly Pro Ala Ala Glu 200 205 210 gaa ctc ttt gat ccg gtt cct gaa cag gat cta ttt gag gcg cta aat 2287Glu Leu Phe Asp Pro Val Pro Glu Gln Asp Leu Phe Glu Ala Leu Asn 215 220 225 gaa acc tta acg cta tgg aac tcg ccg ccc gac tgg gct ggc gat gag 2335Glu Thr Leu Thr Leu Trp Asn Ser Pro Pro Asp Trp Ala Gly Asp Glu 230 235 240 cga aat gta gtg ctt acg ttg tcc cgc att tgg tac agc gca gta acc 2383Arg Asn Val Val Leu Thr Leu Ser Arg Ile Trp Tyr Ser Ala Val Thr 245 250 255 ggc aaa atc gcg ccg aag gat gtc gct gcc gac tgg gca atg gag cgc 2431Gly Lys Ile Ala Pro Lys Asp Val Ala Ala Asp Trp Ala Met Glu Arg 260 265 270 275 ctg ccg gcc cag tat cag ccc gtc ata ctt gaa gct aga cag gct tat 2479Leu Pro Ala Gln Tyr Gln Pro Val Ile Leu Glu Ala Arg Gln Ala Tyr 280 285 290 ctt gga caa gaa gaa gat cgc ttg gcc tcg cgc gca gat cag ttg gaa 2527Leu Gly Gln Glu Glu Asp Arg Leu Ala Ser Arg Ala Asp Gln Leu Glu 295 300 305 gaa ttt gtc cac tac gtg aaa ggc gag atc acc aag gta gtc ggc aaa 2575Glu Phe Val His Tyr Val Lys Gly Glu Ile Thr Lys Val Val Gly Lys 310 315 320 taa tgtctaacaa ttcgttcaag ccgacggata tctagattga tttacgcgcc 2628ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 2688tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 2748cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 2808acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc 2868ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 2928gttccaaact tgaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat 2988tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 3048ttttaacaaa atattaacgt ttacaattta aaaggatcta ggtgaagatc ctttttgata 3108atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 3168aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 3228caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 3288ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 3348cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 3408tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 3468gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 3528ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 3588gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 3648caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 3708ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 3768tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 3828ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 3888agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 3948aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 4008gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac 4068tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga 4128cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 4188cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagcaagg 4248agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag 4308cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg 4368cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga 4428tctgctcatg tttgacagct tatc 445298323PRTEscherichia coli 98Val Val Thr Ala Gln Trp Arg Phe Ser Trp Leu Leu Val Met Thr Cys 1 5 10 15 Phe Phe Gly Val Gln Ser Met Pro Arg Ala Ser Lys Gln Gln Ala Arg 20 25 30 Tyr Ala Val Gly Arg Cys Leu Met Leu Trp Ser Ser Asn Asp Val Thr 35 40 45 Gln Gln Gly Ser Arg Pro Lys Thr Lys Leu Asn Ile Met Arg Glu Ala 50 55 60 Val Ile Ala Glu Val Ser Thr Gln Leu Ser Glu Val Val Gly Val Ile 65 70 75 80 Glu Arg His Leu Glu Pro Thr Leu Leu Ala Val His Leu Tyr Gly Ser 85 90 95 Ala Val Asp Gly Gly Leu Lys Pro His Ser Asp Ile Asp Leu Leu Val 100 105 110 Thr Val Thr Val Arg Leu Asp Glu Thr Thr Arg Arg Ala Leu Ile Asn 115 120 125 Asp Leu Leu Glu Thr Ser Ala Ser Pro Gly Glu Ser Glu Ile Leu Arg 130 135 140 Ala Val Glu Val Thr Ile Val Val His Asp Asp Ile Ile Pro Trp Arg 145 150 155 160 Tyr Pro Ala Lys Arg Glu Leu Gln Phe Gly Glu Trp Gln Arg Asn Asp 165 170 175 Ile Leu Ala Gly Ile Phe Glu Pro Ala Thr Tyr Asp Ile Asp Leu Ala 180 185 190 Ile Leu Leu Thr Lys Ala Arg Glu His Ser Val Ala Leu Val Gly Pro 195 200 205 Ala Ala Glu Glu Leu Phe Asp Pro Val Pro Glu Gln Asp Leu Phe Glu 210 215 220 Ala Leu Asn Glu Thr Leu Thr Leu Trp Asn Ser Pro Pro Asp Trp Ala 225 230 235 240 Gly Asp Glu Arg Asn Val Val Leu Thr Leu Ser Arg Ile Trp Tyr Ser 245 250 255 Ala Val Thr Gly Lys Ile Ala Pro Lys Asp Val Ala Ala Asp Trp Ala 260 265 270 Met Glu Arg Leu Pro Ala Gln Tyr Gln Pro Val Ile Leu Glu Ala Arg 275 280 285 Gln Ala Tyr Leu Gly Gln Glu Glu Asp Arg Leu Ala Ser Arg Ala Asp 290 295 300 Gln Leu Glu Glu Phe Val His Tyr Val Lys Gly Glu Ile Thr Lys Val 305 310 315 320 Val Gly Lys 99903DNAEscherichia coliCDS(1)..(903)NahR_Asn169 gene encoding Salicylic acid biosensor protein 99atg gaa ctg cgt gac ctg gat tta aac ctg ctg gtg gtg ttc aac cag 48Met Glu Leu Arg Asp Leu Asp Leu Asn Leu Leu Val Val Phe Asn Gln 1 5 10 15 ttg ctg gtc gac aga cgc gtc tct atc act gcg gag aac ctg ggc ctg 96Leu Leu Val Asp Arg Arg Val Ser Ile Thr Ala Glu Asn Leu Gly Leu 20 25 30 acc cag cct gcc gtg agc aat gcg ctg aaa cgc ctg cgc acc tcg cta 144Thr Gln Pro Ala Val Ser Asn Ala Leu Lys Arg Leu Arg Thr Ser Leu 35 40 45 cag gac cca ctc ttc gtg cgc aca cat cag gga atg gaa ccc aca ccc 192Gln Asp Pro Leu Phe Val Arg Thr His Gln Gly Met Glu Pro Thr Pro 50 55 60 tat gcc gcg cat ctg gcc gag ccc gtc act tcg gcc atg cac gca ctg 240Tyr Ala Ala His Leu Ala Glu Pro Val Thr Ser Ala Met His Ala Leu 65 70 75 80 cgc aac gcc cta cag cac cat gaa agc ttc gat ccg ctg acc agc gag 288Arg Asn Ala Leu Gln His His Glu Ser Phe Asp Pro Leu Thr Ser Glu 85 90 95 cgt acc ttc acc ctg gcc atg acc gac att ggc gag atc tac ttc atg 336Arg Thr Phe Thr Leu Ala Met Thr Asp Ile Gly Glu Ile Tyr Phe Met 100 105 110 ccg cgg ctg atg gat gtg ctg gct cac cag gcc ccc aat tgc gtg atc 384Pro Arg Leu Met Asp Val Leu Ala His Gln Ala Pro Asn Cys Val Ile 115 120 125 agt acg gtg cgc gac agt tcg atg agc ctg atg cag gcc ttg cag aac 432Ser Thr Val Arg Asp Ser Ser Met Ser Leu Met Gln Ala Leu Gln Asn 130 135 140 gga acc gtg gac ttg gcc gtg ggc ctg ctt ccc aat ctg caa act ggc 480Gly Thr Val Asp Leu Ala Val Gly Leu Leu Pro Asn Leu Gln Thr Gly 145 150 155 160 ttc ttt cag cgc cgg ctg ctc cag aat cac tac gtg tgc cta tgt cgc 528Phe Phe Gln Arg Arg Leu Leu Gln Asn His Tyr Val Cys Leu Cys Arg 165 170 175 aag gac cat cca gtc acc cgc gaa ccc ctg act ctg gag cgc ttc tgt 576Lys Asp His Pro Val Thr Arg Glu Pro Leu Thr Leu Glu Arg Phe Cys 180 185 190 tcc tac ggc cac gtg cgt gtc atc gcc gct ggc acc ggc cac ggc gag 624Ser Tyr Gly His Val Arg Val Ile Ala Ala Gly Thr Gly His Gly Glu 195 200 205 gtg gac acg tac atg aca cgg gtc ggc atc cgg cgc gac atc cgt ctg 672Val Asp Thr Tyr Met Thr Arg Val Gly Ile Arg Arg Asp Ile Arg Leu 210 215 220 gaa gtg ccg cac ttc gcc gcc gtt ggc cac atc ctc cag cgc acc gat 720Glu Val Pro His Phe Ala Ala Val Gly His Ile Leu Gln Arg Thr Asp 225 230 235 240 ctg ctc gcc act gtg ccg ata cgt tta gcc gac tgc tgc gtg gag ccc 768Leu Leu Ala Thr Val Pro Ile Arg Leu Ala Asp Cys Cys Val Glu Pro 245 250 255 ttc ggc cta agc gcc ttg ccg cac cca gtc gtc ttg cct gaa ata gcc 816Phe Gly Leu Ser Ala Leu Pro His Pro Val Val Leu Pro Glu Ile Ala 260 265 270 atc aac atg ttc tgg cat gcg aag tac cac aag gac cta gcc aat att 864Ile Asn Met Phe Trp His Ala Lys Tyr His Lys Asp Leu Ala Asn Ile 275 280 285 tgg ttg cgg caa ctg atg ttt gac ctg ttt acg gat tga 903Trp Leu Arg Gln Leu Met Phe Asp Leu Phe Thr Asp 290 295 300 100300PRTEscherichia coli 100Met Glu Leu Arg Asp Leu Asp Leu Asn Leu Leu Val Val Phe Asn Gln 1 5 10 15 Leu Leu Val Asp Arg Arg Val Ser Ile Thr Ala Glu Asn Leu Gly Leu 20 25 30 Thr Gln Pro Ala Val Ser Asn Ala Leu Lys Arg Leu Arg Thr Ser Leu 35 40 45 Gln Asp Pro Leu Phe Val Arg Thr His Gln Gly Met Glu Pro Thr Pro 50 55 60 Tyr Ala Ala His Leu Ala Glu Pro Val Thr Ser Ala Met His Ala Leu 65 70 75 80 Arg Asn Ala Leu Gln His His Glu Ser Phe Asp Pro Leu Thr Ser Glu 85 90 95 Arg Thr Phe Thr Leu Ala Met Thr Asp Ile Gly Glu Ile Tyr Phe Met 100 105 110 Pro Arg Leu Met Asp Val Leu Ala His Gln Ala Pro Asn Cys Val Ile 115 120 125 Ser Thr Val Arg Asp Ser Ser Met Ser Leu Met Gln Ala Leu Gln Asn 130 135 140 Gly Thr Val Asp Leu Ala Val Gly Leu Leu Pro Asn Leu Gln Thr Gly 145 150 155 160 Phe Phe Gln Arg Arg Leu Leu Gln Asn His Tyr Val Cys Leu Cys Arg 165 170 175 Lys Asp His Pro Val Thr Arg Glu Pro Leu Thr Leu Glu Arg Phe Cys 180 185 190 Ser Tyr Gly His Val Arg Val Ile Ala Ala Gly Thr Gly His Gly Glu 195 200 205 Val Asp Thr Tyr Met Thr Arg Val Gly Ile Arg Arg Asp Ile Arg Leu 210 215 220 Glu Val Pro His Phe Ala Ala Val Gly His Ile Leu Gln Arg Thr Asp 225 230 235 240 Leu Leu Ala Thr Val Pro Ile Arg Leu Ala Asp Cys Cys Val Glu Pro 245 250 255 Phe Gly Leu Ser Ala Leu Pro His Pro Val Val Leu Pro Glu Ile Ala 260 265 270 Ile Asn Met Phe Trp His Ala Lys Tyr His Lys Asp Leu Ala Asn Ile 275 280 285 Trp Leu Arg Gln Leu Met Phe Asp Leu Phe Thr Asp 290 295 300 1012233DNAEscherichia colipromoter(1794)..(1929)pSAL promoterRBS(1930)..(1935)CDS(1941)..(2231)Portion of folp gene 101aagatgctct catgaaatat gagactatcg acgcaccgca gattgatgac ctgatggcac 60gtcgcgatgt acgtccgcca gcgggctggg aagaaccagg cgcttctaac aattctggcg 120acaatggtag tccaaaggct cctcgtccgg ttgatgaacc gcgtacgccg aacccgggta 180acaccatgtc agagcagtta ggcgacaagt aagttcccgc atcagatgac tgtatttgta 240ccgaaaaccc cggggcgtgc tccggggttt tttcttatca attcatacca gggataacat 300cagcgattgt gtaggctgga gctgcttcga agttcctata ctttctagag aataggaact 360tcggaatagg aacttcaaga tccccttatt agaagaactc gtcaagaagg cgatagaagg 420cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt 480cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg 540ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat 600tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct 660tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct 720gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt 780ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga 840tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc 900ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa 960cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac 1020cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg 1080cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc 1140aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc 1200ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca 1260tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg 1320gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag 1380ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat 1440tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc 1500agcccttgcg ccctgagtgc ttgcggcagc gtgagcttca aaagcgctct gaagttccta 1560tactttctag agaataggaa cttcgaactg caggtcgacg gatccccgga atattcatat 1620gtctatgtgc gcacgaagag tgggtcctgt agcgaggtgc gcaggcgttt cagcgcattg 1680ctcacggcag gctgggtcag gcccaggttc tccgcagtga tagagacgcg tctgtcgacc 1740agcaactggt tgaacaccac cagcaggttt aaatccaggt cacgcagttc catggggcct 1800cgcttgggtt attgctggtg cccggccggg cgcaatattc atgttgatga tttattatat 1860atcgagtggt gtatttatca atattgtttg ctccgttatc gttattaaca agtcatcaat 1920aaagccatca ggagtaaggt atg aaa ctc ttt gcc cag ggt act tca ctg gac 1973 Met Lys Leu Phe Ala Gln Gly Thr Ser Leu Asp 1 5 10 ctt agc cat cct cac gta atg ggg atc ctc aac gtc acg cct gat tcc 2021Leu Ser His Pro His Val Met Gly Ile Leu Asn Val Thr Pro Asp Ser 15 20 25 ttt tcg gat ggt ggc acg cat aac tcg ctg ata gat gcg gtg aaa cat 2069Phe Ser Asp Gly Gly Thr His Asn Ser Leu Ile Asp Ala Val Lys His 30 35 40 gcg aat ctg atg atc aac gct ggc gcg acg atc att gac gtt ggt ggc 2117Ala Asn Leu Met Ile Asn Ala Gly Ala Thr Ile Ile Asp Val Gly Gly

45 50 55 gag tcc acg cgc cca ggg gcg gcg gaa gtt agc gtt gaa gaa gag ttg 2165Glu Ser Thr Arg Pro Gly Ala Ala Glu Val Ser Val Glu Glu Glu Leu 60 65 70 75 caa cgt gtt att cct gtg gtt gag gca att gct caa cgc ttc gaa gtc 2213Gln Arg Val Ile Pro Val Val Glu Ala Ile Ala Gln Arg Phe Glu Val 80 85 90 tgg atc tca gtc gat aca tc 2233Trp Ile Ser Val Asp Thr 95 10297PRTEscherichia coli 102Met Lys Leu Phe Ala Gln Gly Thr Ser Leu Asp Leu Ser His Pro His 1 5 10 15 Val Met Gly Ile Leu Asn Val Thr Pro Asp Ser Phe Ser Asp Gly Gly 20 25 30 Thr His Asn Ser Leu Ile Asp Ala Val Lys His Ala Asn Leu Met Ile 35 40 45 Asn Ala Gly Ala Thr Ile Ile Asp Val Gly Gly Glu Ser Thr Arg Pro 50 55 60 Gly Ala Ala Glu Val Ser Val Glu Glu Glu Leu Gln Arg Val Ile Pro 65 70 75 80 Val Val Glu Ala Ile Ala Gln Arg Phe Glu Val Trp Ile Ser Val Asp 85 90 95 Thr 1034452DNAEscherichia coliCDS(1607)..(2578)SpecR gene in pBABspec 103atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accactcaat ttcaatccgt aaacaggtca 120aacatcagtt gccgcaacca aatattggct aggtccttgt ggtacttcgc atgccagaac 180atgttgatgg ctatttcagg caagacgact gggtgcggca aggcgcttag gccgaagggc 240tccacgcagc agtcggctaa acgtatcggc acagtggcga gcagatcggt gcgctggagg 300atgtggccaa cggcggcgaa gtgcggcact tccagacgga tgtcgcgccg gatgccgacc 360cgtgtcatgt acgtgtccac ctcgccgtgg ccggtgccag cggcgatgac acgcacgtgg 420ccgtaggaac agaagcgctc cagagtcagg ggttcgcggg tgactggatg gtccttgcga 480cataggcaca cgtagtgatc ctggagcagc cggcgctgaa agaagccagt ttgcagattg 540ggaagcaggc ccacggccaa gtccacggtt ccgttctgca aggcctgcat caggctcatc 600gaactgtcgc gcaccgtact gatcacgcaa ttgggggcct ggtgagccag cacatccatc 660agccgcggca tgaagtagat ctcgccaatg tcggtcatgg ccagggtgaa ggtacgctcg 720ctggtcagcg gatcgaagct ttcatggtgc tgtagggcgt tgcgcagtgc gtgcatggcc 780gaagtgacgg gctcggccag atgcgcggca tagggtgtgg gttccattcc ctgatgtgtg 840cgcacgaaga gtgggtcctg tagcgaggtg cgcaggcgtt tcagcgcatt gctcacggca 900ggctgggtca ggcccaggtt ctccgcagtg atagagacgc gtctgtcgac cagcaactgg 960ttgaacacca ccagcaggtt taaatccagg tcacgcagtt ccatggggcc tcgcttgggt 1020tgctagcatt gtacctagga ctgagctagc cgtaaatcct ctagagtcga cctgcaggca 1080tgcaagcttg gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca 1140gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca 1200cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct 1260ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga 1320ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc 1380gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc 1440gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggtga tatccggatg 1500aaggcacgaa cccagtggac ataagcctcg ttcggttcgt aagctgtaat gcaagtagcg 1560taactgccgt cacgcaactg gtccacaacc ttgaccgaac gcagcg gtg gta acg 1615 Val Val Thr 1 gcg cag tgg cgg ttt tca tgg ctt ctt gtt atg aca tgt ttt ttt ggg 1663Ala Gln Trp Arg Phe Ser Trp Leu Leu Val Met Thr Cys Phe Phe Gly 5 10 15 gta cag tct atg cct cgg gca tcc aag cag caa gcg cgt tac gcc gtg 1711Val Gln Ser Met Pro Arg Ala Ser Lys Gln Gln Ala Arg Tyr Ala Val 20 25 30 35 ggt cga tgt ttg atg tta tgg agc agc aac gat gtt acg cag cag ggc 1759Gly Arg Cys Leu Met Leu Trp Ser Ser Asn Asp Val Thr Gln Gln Gly 40 45 50 agt cgc cct aaa aca aag tta aac atc atg agg gaa gcg gtg atc gcc 1807Ser Arg Pro Lys Thr Lys Leu Asn Ile Met Arg Glu Ala Val Ile Ala 55 60 65 gaa gta tcg act caa cta tca gag gta gtt ggc gtc atc gag cgc cat 1855Glu Val Ser Thr Gln Leu Ser Glu Val Val Gly Val Ile Glu Arg His 70 75 80 ctc gaa ccg acg ttg ctg gcc gta cat ttg tac ggc tcc gca gtg gat 1903Leu Glu Pro Thr Leu Leu Ala Val His Leu Tyr Gly Ser Ala Val Asp 85 90 95 ggc ggc ctg aag cca cac agt gat att gat ttg ctg gtt acg gtg acc 1951Gly Gly Leu Lys Pro His Ser Asp Ile Asp Leu Leu Val Thr Val Thr 100 105 110 115 gta agg ctt gat gaa aca acg cgg cga gct ttg atc aac gac ctt ttg 1999Val Arg Leu Asp Glu Thr Thr Arg Arg Ala Leu Ile Asn Asp Leu Leu 120 125 130 gaa act tcg gct tcc cct gga gag agc gag att ctc cgc gct gta gaa 2047Glu Thr Ser Ala Ser Pro Gly Glu Ser Glu Ile Leu Arg Ala Val Glu 135 140 145 gtc acc att gtt gtg cac gac gac atc att ccg tgg cgt tat cca gct 2095Val Thr Ile Val Val His Asp Asp Ile Ile Pro Trp Arg Tyr Pro Ala 150 155 160 aag cgc gaa ctg caa ttt gga gaa tgg cag cgc aat gac att ctt gca 2143Lys Arg Glu Leu Gln Phe Gly Glu Trp Gln Arg Asn Asp Ile Leu Ala 165 170 175 ggt atc ttc gag cca gcc acg tac gac att gat ctg gct atc ttg ctg 2191Gly Ile Phe Glu Pro Ala Thr Tyr Asp Ile Asp Leu Ala Ile Leu Leu 180 185 190 195 aca aaa gca aga gaa cat agc gtt gcc ttg gta ggt cca gcg gcg gag 2239Thr Lys Ala Arg Glu His Ser Val Ala Leu Val Gly Pro Ala Ala Glu 200 205 210 gaa ctc ttt gat ccg gtt cct gaa cag gat cta ttt gag gcg cta aat 2287Glu Leu Phe Asp Pro Val Pro Glu Gln Asp Leu Phe Glu Ala Leu Asn 215 220 225 gaa acc tta acg cta tgg aac tcg ccg ccc gac tgg gct ggc gat gag 2335Glu Thr Leu Thr Leu Trp Asn Ser Pro Pro Asp Trp Ala Gly Asp Glu 230 235 240 cga aat gta gtg ctt acg ttg tcc cgc att tgg tac agc gca gta acc 2383Arg Asn Val Val Leu Thr Leu Ser Arg Ile Trp Tyr Ser Ala Val Thr 245 250 255 ggc aaa atc gcg ccg aag gat gtc gct gcc gac tgg gca atg gag cgc 2431Gly Lys Ile Ala Pro Lys Asp Val Ala Ala Asp Trp Ala Met Glu Arg 260 265 270 275 ctg ccg gcc cag tat cag ccc gtc ata ctt gaa gct aga cag gct tat 2479Leu Pro Ala Gln Tyr Gln Pro Val Ile Leu Glu Ala Arg Gln Ala Tyr 280 285 290 ctt gga caa gaa gaa gat cgc ttg gcc tcg cgc gca gat cag ttg gaa 2527Leu Gly Gln Glu Glu Asp Arg Leu Ala Ser Arg Ala Asp Gln Leu Glu 295 300 305 gaa ttt gtc cac tac gtg aaa ggc gag atc acc aag gta gtc ggc aaa 2575Glu Phe Val His Tyr Val Lys Gly Glu Ile Thr Lys Val Val Gly Lys 310 315 320 taa tgtctaacaa ttcgttcaag ccgacggata tctagattga tttacgcgcc 2628ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 2688tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 2748cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 2808acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc 2868ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 2928gttccaaact tgaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat 2988tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 3048ttttaacaaa atattaacgt ttacaattta aaaggatcta ggtgaagatc ctttttgata 3108atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 3168aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 3228caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 3288ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 3348cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 3408tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 3468gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 3528ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 3588gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 3648caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 3708ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 3768tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 3828ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 3888agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 3948aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 4008gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac 4068tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga 4128cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 4188cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagcaagg 4248agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag 4308cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg 4368cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga 4428tctgctcatg tttgacagct tatc 4452104323PRTEscherichia coli 104Val Val Thr Ala Gln Trp Arg Phe Ser Trp Leu Leu Val Met Thr Cys 1 5 10 15 Phe Phe Gly Val Gln Ser Met Pro Arg Ala Ser Lys Gln Gln Ala Arg 20 25 30 Tyr Ala Val Gly Arg Cys Leu Met Leu Trp Ser Ser Asn Asp Val Thr 35 40 45 Gln Gln Gly Ser Arg Pro Lys Thr Lys Leu Asn Ile Met Arg Glu Ala 50 55 60 Val Ile Ala Glu Val Ser Thr Gln Leu Ser Glu Val Val Gly Val Ile 65 70 75 80 Glu Arg His Leu Glu Pro Thr Leu Leu Ala Val His Leu Tyr Gly Ser 85 90 95 Ala Val Asp Gly Gly Leu Lys Pro His Ser Asp Ile Asp Leu Leu Val 100 105 110 Thr Val Thr Val Arg Leu Asp Glu Thr Thr Arg Arg Ala Leu Ile Asn 115 120 125 Asp Leu Leu Glu Thr Ser Ala Ser Pro Gly Glu Ser Glu Ile Leu Arg 130 135 140 Ala Val Glu Val Thr Ile Val Val His Asp Asp Ile Ile Pro Trp Arg 145 150 155 160 Tyr Pro Ala Lys Arg Glu Leu Gln Phe Gly Glu Trp Gln Arg Asn Asp 165 170 175 Ile Leu Ala Gly Ile Phe Glu Pro Ala Thr Tyr Asp Ile Asp Leu Ala 180 185 190 Ile Leu Leu Thr Lys Ala Arg Glu His Ser Val Ala Leu Val Gly Pro 195 200 205 Ala Ala Glu Glu Leu Phe Asp Pro Val Pro Glu Gln Asp Leu Phe Glu 210 215 220 Ala Leu Asn Glu Thr Leu Thr Leu Trp Asn Ser Pro Pro Asp Trp Ala 225 230 235 240 Gly Asp Glu Arg Asn Val Val Leu Thr Leu Ser Arg Ile Trp Tyr Ser 245 250 255 Ala Val Thr Gly Lys Ile Ala Pro Lys Asp Val Ala Ala Asp Trp Ala 260 265 270 Met Glu Arg Leu Pro Ala Gln Tyr Gln Pro Val Ile Leu Glu Ala Arg 275 280 285 Gln Ala Tyr Leu Gly Gln Glu Glu Asp Arg Leu Ala Ser Arg Ala Asp 290 295 300 Gln Leu Glu Glu Phe Val His Tyr Val Lys Gly Glu Ile Thr Lys Val 305 310 315 320 Val Gly Lys 105903DNAEscherichia coliCDS(1)..(903)NahR gene encoding NahR benzoic acid biosensor 105atg gaa ctg cgt gac ctg gat tta aac ctg ctg gtg gtg ttc aac cag 48Met Glu Leu Arg Asp Leu Asp Leu Asn Leu Leu Val Val Phe Asn Gln 1 5 10 15 ttg ctg gtc gac aga cgc gtc tct atc act gcg gag aac ctg ggc ctg 96Leu Leu Val Asp Arg Arg Val Ser Ile Thr Ala Glu Asn Leu Gly Leu 20 25 30 acc cag cct gcc gtg agc aat gcg ctg aaa cgc ctg cgc acc tcg cta 144Thr Gln Pro Ala Val Ser Asn Ala Leu Lys Arg Leu Arg Thr Ser Leu 35 40 45 cag gac cca ctc ttc gtg cgc aca cat cag gga atg gaa ccc aca ccc 192Gln Asp Pro Leu Phe Val Arg Thr His Gln Gly Met Glu Pro Thr Pro 50 55 60 tat gcc gcg cat ctg gcc gag ccc gtc act tcg gcc atg cac gca ctg 240Tyr Ala Ala His Leu Ala Glu Pro Val Thr Ser Ala Met His Ala Leu 65 70 75 80 cgc aac gcc cta cag cac cat gaa agc ttc gat ccg ctg acc agc gag 288Arg Asn Ala Leu Gln His His Glu Ser Phe Asp Pro Leu Thr Ser Glu 85 90 95 cgt acc ttc acc ctg gcc atg acc gac att ggc gag atc tac ttc atg 336Arg Thr Phe Thr Leu Ala Met Thr Asp Ile Gly Glu Ile Tyr Phe Met 100 105 110 ccg cgg ctg atg gat gtg ctg gct cac cag gcc ccc aat tgc gtg atc 384Pro Arg Leu Met Asp Val Leu Ala His Gln Ala Pro Asn Cys Val Ile 115 120 125 agt acg gtg cgc gac agt tcg atg agc ctg atg cag gcc ttg cag aac 432Ser Thr Val Arg Asp Ser Ser Met Ser Leu Met Gln Ala Leu Gln Asn 130 135 140 gga acc gtg gac ttg gcc gtg ggc ctg ctt ccc aat ctg caa act ggc 480Gly Thr Val Asp Leu Ala Val Gly Leu Leu Pro Asn Leu Gln Thr Gly 145 150 155 160 ttc ttt cag cgc cgg ctg ctc cag gat cac tac gtg tgc cta tgt cgc 528Phe Phe Gln Arg Arg Leu Leu Gln Asp His Tyr Val Cys Leu Cys Arg 165 170 175 aag gac cat cca gtc acc cgc gaa ccc ctg act ctg gag cgc ttc tgt 576Lys Asp His Pro Val Thr Arg Glu Pro Leu Thr Leu Glu Arg Phe Cys 180 185 190 tcc tac ggc cac gtg cgt gtc atc gcc gct ggc acc ggc cac ggc gag 624Ser Tyr Gly His Val Arg Val Ile Ala Ala Gly Thr Gly His Gly Glu 195 200 205 gtg gac acg tac atg aca cgg gtc ggc atc cgg cgc gac atc cgt ctg 672Val Asp Thr Tyr Met Thr Arg Val Gly Ile Arg Arg Asp Ile Arg Leu 210 215 220 gaa gtg ccg cac ttc gcc gcc gtt ggc cac atc ctc cag cgc acc gat 720Glu Val Pro His Phe Ala Ala Val Gly His Ile Leu Gln Arg Thr Asp 225 230 235 240 ctg ctc gcc act gtg ccg ata cgt tta gcc gac tgc tgc gtg gag ccc 768Leu Leu Ala Thr Val Pro Ile Arg Leu Ala Asp Cys Cys Val Glu Pro 245 250 255 ttc ggc cta agc gcc ttg ccg cac cca gtc gtc ttg cct gaa ata gcc 816Phe Gly Leu Ser Ala Leu Pro His Pro Val Val Leu Pro Glu Ile Ala 260 265 270 atc aac atg ttc tgg cat gcg aag tac cac aag gac cta gcc aat att 864Ile Asn Met Phe Trp His Ala Lys Tyr His Lys Asp Leu Ala Asn Ile 275 280 285 tgg ttg cgg caa ctg atg ttt gac ctg ttt acg gat tga 903Trp Leu Arg Gln Leu Met Phe Asp Leu Phe Thr Asp 290 295 300 106300PRTEscherichia coli 106Met Glu Leu Arg Asp Leu Asp Leu Asn Leu Leu Val Val Phe Asn Gln 1 5 10 15 Leu Leu Val Asp Arg Arg Val Ser Ile Thr Ala Glu Asn Leu Gly Leu 20 25 30 Thr Gln Pro Ala Val Ser Asn Ala Leu Lys Arg Leu Arg Thr Ser Leu 35 40 45 Gln Asp Pro Leu Phe Val Arg Thr His Gln Gly Met Glu Pro Thr Pro 50 55 60 Tyr Ala Ala His Leu Ala Glu Pro Val Thr Ser Ala Met His Ala Leu 65 70 75 80 Arg Asn Ala Leu Gln His His Glu Ser Phe Asp Pro Leu Thr Ser Glu 85 90 95 Arg Thr Phe Thr Leu Ala Met Thr Asp Ile Gly Glu Ile Tyr Phe Met 100 105 110 Pro Arg Leu Met Asp Val Leu Ala His Gln Ala Pro Asn Cys Val Ile 115 120 125 Ser Thr Val Arg Asp Ser Ser Met

Ser Leu Met Gln Ala Leu Gln Asn 130 135 140 Gly Thr Val Asp Leu Ala Val Gly Leu Leu Pro Asn Leu Gln Thr Gly 145 150 155 160 Phe Phe Gln Arg Arg Leu Leu Gln Asp His Tyr Val Cys Leu Cys Arg 165 170 175 Lys Asp His Pro Val Thr Arg Glu Pro Leu Thr Leu Glu Arg Phe Cys 180 185 190 Ser Tyr Gly His Val Arg Val Ile Ala Ala Gly Thr Gly His Gly Glu 195 200 205 Val Asp Thr Tyr Met Thr Arg Val Gly Ile Arg Arg Asp Ile Arg Leu 210 215 220 Glu Val Pro His Phe Ala Ala Val Gly His Ile Leu Gln Arg Thr Asp 225 230 235 240 Leu Leu Ala Thr Val Pro Ile Arg Leu Ala Asp Cys Cys Val Glu Pro 245 250 255 Phe Gly Leu Ser Ala Leu Pro His Pro Val Val Leu Pro Glu Ile Ala 260 265 270 Ile Asn Met Phe Trp His Ala Lys Tyr His Lys Asp Leu Ala Asn Ile 275 280 285 Trp Leu Arg Gln Leu Met Phe Asp Leu Phe Thr Asp 290 295 300 107175DNAEscherichia colipromoter(1)..(175)dmpRKLM promoter 107gcatttgctc aagcggcctt gggcaattga tcaaatgctt aaaaagtctg cgcaagcgcg 60gcttaatttc gctcgctccg atcattctaa aaattagaaa cacattgaaa aacattacct 120tgaagtctgt tttcagacct tggcacagcc gttgcttgat gtcctgcgta ctaga 1751081692DNAEscherichia coliCDS(1)..(1692)DmpR gene encoding DmpR biosensor 108atg ccg atc aag tac aag cct gaa atc cag cac tcc gat ttc aag gac 48Met Pro Ile Lys Tyr Lys Pro Glu Ile Gln His Ser Asp Phe Lys Asp 1 5 10 15 ctg acc aac ctg atc cac ttc cag agc atg gaa ggc aag atc tgg ctt 96Leu Thr Asn Leu Ile His Phe Gln Ser Met Glu Gly Lys Ile Trp Leu 20 25 30 ggc gaa cag cgc atg ctg ttg ctt cag ttt tca gcg atg gcc agc ttt 144Gly Glu Gln Arg Met Leu Leu Leu Gln Phe Ser Ala Met Ala Ser Phe 35 40 45 cgc cgg gaa atg gtc aat acc ctg ggc atc gaa cgc gcc aag ggc ttg 192Arg Arg Glu Met Val Asn Thr Leu Gly Ile Glu Arg Ala Lys Gly Leu 50 55 60 ttc ctg cgc cat ggt tac cag tcc ggc ctg aag gat gcc gaa ctg gcc 240Phe Leu Arg His Gly Tyr Gln Ser Gly Leu Lys Asp Ala Glu Leu Ala 65 70 75 80 agg aag ctg aga ccg aat gcc agc gaa gtc ggc atg ttc ctc gct ggg 288Arg Lys Leu Arg Pro Asn Ala Ser Glu Val Gly Met Phe Leu Ala Gly 85 90 95 ccg cag atg cat tca ctc aag ggt ctg gtc aag gtc cgc ccc acc gag 336Pro Gln Met His Ser Leu Lys Gly Leu Val Lys Val Arg Pro Thr Glu 100 105 110 ctc gat atc gac aag gaa tac ggg cgc ttc tat gcc gag atg gag tgg 384Leu Asp Ile Asp Lys Glu Tyr Gly Arg Phe Tyr Ala Glu Met Glu Trp 115 120 125 atc gac tcc ttc gag gtg gaa atc tgc cag acc gac ctg ggg cag atg 432Ile Asp Ser Phe Glu Val Glu Ile Cys Gln Thr Asp Leu Gly Gln Met 130 135 140 caa gac ccg gtg tgc tgg act ctg ctc ggc tac gcc tgc gcc tat tcc 480Gln Asp Pro Val Cys Trp Thr Leu Leu Gly Tyr Ala Cys Ala Tyr Ser 145 150 155 160 tcg gcg ttc atg ggc cgg gaa atc atc ttc aag gaa gtc agc tgc ggc 528Ser Ala Phe Met Gly Arg Glu Ile Ile Phe Lys Glu Val Ser Cys Gly 165 170 175 ggc tgc ggc ggc gac aag tgc cgg gtc att ggc aag ccg gcc gaa gag 576Gly Cys Gly Gly Asp Lys Cys Arg Val Ile Gly Lys Pro Ala Glu Glu 180 185 190 tgg gac gac gtt gcc agc ttc aaa cag tat ttc aag aac gac ccc atc 624Trp Asp Asp Val Ala Ser Phe Lys Gln Tyr Phe Lys Asn Asp Pro Ile 195 200 205 atc gag gaa ctc tac gag ttg caa tcg caa ctg ttg tcg ctg cgt acc 672Ile Glu Glu Leu Tyr Glu Leu Gln Ser Gln Leu Leu Ser Leu Arg Thr 210 215 220 aac ctc gac aaa cag gaa ggc cag tac tac ggc atc ggt cag acc ccg 720Asn Leu Asp Lys Gln Glu Gly Gln Tyr Tyr Gly Ile Gly Gln Thr Pro 225 230 235 240 gcc tac cag acc gtg cgc aat atg atg gac aag gcc gca cag ggc aaa 768Ala Tyr Gln Thr Val Arg Asn Met Met Asp Lys Ala Ala Gln Gly Lys 245 250 255 gtc tcg gtg ctg ctg ctt ggc gag acc ggg gtc ggc aag gag gtc atc 816Val Ser Val Leu Leu Leu Gly Glu Thr Gly Val Gly Lys Glu Val Ile 260 265 270 gcg cgt agc gtg cac ctg cgc agc aaa cgc gcc gcc gag ccc ttt gtc 864Ala Arg Ser Val His Leu Arg Ser Lys Arg Ala Ala Glu Pro Phe Val 275 280 285 gcg gtg aac tgt gcg gcg atc ccg ccg gac ctg atc gag tcc gaa ttg 912Ala Val Asn Cys Ala Ala Ile Pro Pro Asp Leu Ile Glu Ser Glu Leu 290 295 300 ttc ggc gtg gaa aaa ggc gcc ttc acc ggc gcc tcc cag tca cgc atg 960Phe Gly Val Glu Lys Gly Ala Phe Thr Gly Ala Ser Gln Ser Arg Met 305 310 315 320 ggc cgc ttc gag cgg gcc gac aag ggc acc atc ttc ctt gac gag gtg 1008Gly Arg Phe Glu Arg Ala Asp Lys Gly Thr Ile Phe Leu Asp Glu Val 325 330 335 atc gaa ctc agc ccg cgc gct cag gcc agt ctg ctg cgc gtg ctg caa 1056Ile Glu Leu Ser Pro Arg Ala Gln Ala Ser Leu Leu Arg Val Leu Gln 340 345 350 gaa ggc gag ctg gag cga gtt ggc gac aac cgc acg cgc aag atc gac 1104Glu Gly Glu Leu Glu Arg Val Gly Asp Asn Arg Thr Arg Lys Ile Asp 355 360 365 gta agg gtt atc gcc gcc acc cac gag gac ctg gcc gaa gcg gtc aag 1152Val Arg Val Ile Ala Ala Thr His Glu Asp Leu Ala Glu Ala Val Lys 370 375 380 gcc ggg cgt ttt cgc gcc gac ctg tac tac cga ctg aac gtt ttc ccg 1200Ala Gly Arg Phe Arg Ala Asp Leu Tyr Tyr Arg Leu Asn Val Phe Pro 385 390 395 400 gtg gcg atc ccg gcg ttg cgc gaa cgc cgc gag gac att cca ctg ctg 1248Val Ala Ile Pro Ala Leu Arg Glu Arg Arg Glu Asp Ile Pro Leu Leu 405 410 415 gtt gag cac ttc ctt cag cgc ttc cac cag gag tac ggc aag aga acc 1296Val Glu His Phe Leu Gln Arg Phe His Gln Glu Tyr Gly Lys Arg Thr 420 425 430 ctc ggc ctt tca gac aaa gcc ctg gag gcc tgc ctg cat tac agt tgg 1344Leu Gly Leu Ser Asp Lys Ala Leu Glu Ala Cys Leu His Tyr Ser Trp 435 440 445 ccg ggc aat atc cgt gag ctg gag aac gtc atc gag cgc ggc atc atc 1392Pro Gly Asn Ile Arg Glu Leu Glu Asn Val Ile Glu Arg Gly Ile Ile 450 455 460 ctc acc gat ccg aac gaa agc atc agc gtg cag gcg ctg ttc cta cgg 1440Leu Thr Asp Pro Asn Glu Ser Ile Ser Val Gln Ala Leu Phe Leu Arg 465 470 475 480 gcg ccg gaa gag ccg cag acc gcc agc gag cgg gtg tcg ttg gac ggc 1488Ala Pro Glu Glu Pro Gln Thr Ala Ser Glu Arg Val Ser Leu Asp Gly 485 490 495 gtg ctg att cag cca ggc aat ggc cag ggc agt tgg atc agc cag ttg 1536Val Leu Ile Gln Pro Gly Asn Gly Gln Gly Ser Trp Ile Ser Gln Leu 500 505 510 ttg agc agc ggc ctg agc ctc gac gag atc gag gaa agc ctg atg cgc 1584Leu Ser Ser Gly Leu Ser Leu Asp Glu Ile Glu Glu Ser Leu Met Arg 515 520 525 gaa gcc atg caa cag gcc aac caa aac gtc tcc ggt gcc gcg cgc ttg 1632Glu Ala Met Gln Gln Ala Asn Gln Asn Val Ser Gly Ala Ala Arg Leu 530 535 540 ctc ggc cta agc cga ccg gca ctg gcc tat cgg ctg aag aaa atc ggc 1680Leu Gly Leu Ser Arg Pro Ala Leu Ala Tyr Arg Leu Lys Lys Ile Gly 545 550 555 560 atc gaa ggc tag 1692Ile Glu Gly 109563PRTEscherichia coli 109Met Pro Ile Lys Tyr Lys Pro Glu Ile Gln His Ser Asp Phe Lys Asp 1 5 10 15 Leu Thr Asn Leu Ile His Phe Gln Ser Met Glu Gly Lys Ile Trp Leu 20 25 30 Gly Glu Gln Arg Met Leu Leu Leu Gln Phe Ser Ala Met Ala Ser Phe 35 40 45 Arg Arg Glu Met Val Asn Thr Leu Gly Ile Glu Arg Ala Lys Gly Leu 50 55 60 Phe Leu Arg His Gly Tyr Gln Ser Gly Leu Lys Asp Ala Glu Leu Ala 65 70 75 80 Arg Lys Leu Arg Pro Asn Ala Ser Glu Val Gly Met Phe Leu Ala Gly 85 90 95 Pro Gln Met His Ser Leu Lys Gly Leu Val Lys Val Arg Pro Thr Glu 100 105 110 Leu Asp Ile Asp Lys Glu Tyr Gly Arg Phe Tyr Ala Glu Met Glu Trp 115 120 125 Ile Asp Ser Phe Glu Val Glu Ile Cys Gln Thr Asp Leu Gly Gln Met 130 135 140 Gln Asp Pro Val Cys Trp Thr Leu Leu Gly Tyr Ala Cys Ala Tyr Ser 145 150 155 160 Ser Ala Phe Met Gly Arg Glu Ile Ile Phe Lys Glu Val Ser Cys Gly 165 170 175 Gly Cys Gly Gly Asp Lys Cys Arg Val Ile Gly Lys Pro Ala Glu Glu 180 185 190 Trp Asp Asp Val Ala Ser Phe Lys Gln Tyr Phe Lys Asn Asp Pro Ile 195 200 205 Ile Glu Glu Leu Tyr Glu Leu Gln Ser Gln Leu Leu Ser Leu Arg Thr 210 215 220 Asn Leu Asp Lys Gln Glu Gly Gln Tyr Tyr Gly Ile Gly Gln Thr Pro 225 230 235 240 Ala Tyr Gln Thr Val Arg Asn Met Met Asp Lys Ala Ala Gln Gly Lys 245 250 255 Val Ser Val Leu Leu Leu Gly Glu Thr Gly Val Gly Lys Glu Val Ile 260 265 270 Ala Arg Ser Val His Leu Arg Ser Lys Arg Ala Ala Glu Pro Phe Val 275 280 285 Ala Val Asn Cys Ala Ala Ile Pro Pro Asp Leu Ile Glu Ser Glu Leu 290 295 300 Phe Gly Val Glu Lys Gly Ala Phe Thr Gly Ala Ser Gln Ser Arg Met 305 310 315 320 Gly Arg Phe Glu Arg Ala Asp Lys Gly Thr Ile Phe Leu Asp Glu Val 325 330 335 Ile Glu Leu Ser Pro Arg Ala Gln Ala Ser Leu Leu Arg Val Leu Gln 340 345 350 Glu Gly Glu Leu Glu Arg Val Gly Asp Asn Arg Thr Arg Lys Ile Asp 355 360 365 Val Arg Val Ile Ala Ala Thr His Glu Asp Leu Ala Glu Ala Val Lys 370 375 380 Ala Gly Arg Phe Arg Ala Asp Leu Tyr Tyr Arg Leu Asn Val Phe Pro 385 390 395 400 Val Ala Ile Pro Ala Leu Arg Glu Arg Arg Glu Asp Ile Pro Leu Leu 405 410 415 Val Glu His Phe Leu Gln Arg Phe His Gln Glu Tyr Gly Lys Arg Thr 420 425 430 Leu Gly Leu Ser Asp Lys Ala Leu Glu Ala Cys Leu His Tyr Ser Trp 435 440 445 Pro Gly Asn Ile Arg Glu Leu Glu Asn Val Ile Glu Arg Gly Ile Ile 450 455 460 Leu Thr Asp Pro Asn Glu Ser Ile Ser Val Gln Ala Leu Phe Leu Arg 465 470 475 480 Ala Pro Glu Glu Pro Gln Thr Ala Ser Glu Arg Val Ser Leu Asp Gly 485 490 495 Val Leu Ile Gln Pro Gly Asn Gly Gln Gly Ser Trp Ile Ser Gln Leu 500 505 510 Leu Ser Ser Gly Leu Ser Leu Asp Glu Ile Glu Glu Ser Leu Met Arg 515 520 525 Glu Ala Met Gln Gln Ala Asn Gln Asn Val Ser Gly Ala Ala Arg Leu 530 535 540 Leu Gly Leu Ser Arg Pro Ala Leu Ala Tyr Arg Leu Lys Lys Ile Gly 545 550 555 560 Ile Glu Gly 110589DNAEscherichia colipromoter(1)..(589)hbpC promoter 110gtggtggtgg gattcagaat atggtgaaac cagctcgtcc gagcaggcgc attctggcat 60ctgccgacgg atccatattc ccttcagtct tactgtgtgt aggtgcatgg atcgggctgt 120cgcctagcca gatgcgtcct tcgttgtggg gagggaatgc agaccgaacg ccgaaacatg 180gatttcaggg cgcgatagat cggcaacgat ggtgcggttt tcatggttct tatttttgtt 240agatttcatg gtgatagctc atgccaggta ggagtagata gggttctcgt agatttaata 300aatttatgaa atcgtggttg tgagttttca taatatggtg aagcttgccc gccatggcaa 360gtgcatttcg gcagctgttg gcgaccgcgg aaggggttta cagccgtctg gagctaggct 420ttctggcgct cattaaaata aaaatcctta taaaacagta tcctagcttt tatgtctgag 480gctgcttagt caacctggca cggtactggc tacgagtccc gcaaggcagc cgtgagttcg 540actgtactgc tgcctattaa aacaatatga acgcggagac gtgataaca 5891111713DNAEscherichia coliCDS(1)..(1713)HbpR gene encoding HbpR biosensor 111atg aaa tca aat aaa aat aat agc gac gat cgc tcc atc gtt gcc gac 48Met Lys Ser Asn Lys Asn Asn Ser Asp Asp Arg Ser Ile Val Ala Asp 1 5 10 15 ctt gcg ctc ccc gaa gtc cat gct tta gtt tcc aag ctg cac ttt tct 96Leu Ala Leu Pro Glu Val His Ala Leu Val Ser Lys Leu His Phe Ser 20 25 30 ccc aac gaa gga cgt atc tgg cta gac gaa agc cga tgc ctg ctc cta 144Pro Asn Glu Gly Arg Ile Trp Leu Asp Glu Ser Arg Cys Leu Leu Leu 35 40 45 caa gtg gag aca ctg aag gat ata tac aag gaa ttg cag gcc tat tct 192Gln Val Glu Thr Leu Lys Asp Ile Tyr Lys Glu Leu Gln Ala Tyr Ser 50 55 60 ggc cca gac tac acg cgg gag ttt ttg act cgt atc ggc ttc acc acc 240Gly Pro Asp Tyr Thr Arg Glu Phe Leu Thr Arg Ile Gly Phe Thr Thr 65 70 75 80 ggc caa cgc gat gca gaa atg att ata aaa aaa caa ggt ata tcc tct 288Gly Gln Arg Asp Ala Glu Met Ile Ile Lys Lys Gln Gly Ile Ser Ser 85 90 95 atc aaa gaa cag att tat gca ggt ggt gtc ttg cac gcc ttg caa ggg 336Ile Lys Glu Gln Ile Tyr Ala Gly Gly Val Leu His Ala Leu Gln Gly 100 105 110 ttt cta aca tct ata caa gct gga tca tcg gcg cta aat gcg gtc gat 384Phe Leu Thr Ser Ile Gln Ala Gly Ser Ser Ala Leu Asn Ala Val Asp 115 120 125 atg aag agc atg gat tat cat gct gag gcc tac tgg cag aac tct atc 432Met Lys Ser Met Asp Tyr His Ala Glu Ala Tyr Trp Gln Asn Ser Ile 130 135 140 gaa gct gaa att cac tta gcg atg cat ggt gtc agt tca cat gcg gta 480Glu Ala Glu Ile His Leu Ala Met His Gly Val Ser Ser His Ala Val 145 150 155 160 tgt tgg ttt tcc gtt gcg tat tgc tca ggg tac cta agc gca tgc gct 528Cys Trp Phe Ser Val Ala Tyr Cys Ser Gly Tyr Leu Ser Ala Cys Ala 165 170 175 gga aaa ccc att gtc gtg gaa gag atc gaa tgc caa gcg atg gga cac 576Gly Lys Pro Ile Val Val Glu Glu Ile Glu Cys Gln Ala Met Gly His 180 185 190 act cat tgc cgt att caa gcg aag ccc gcc gaa atg tgg gcg ctc agt 624Thr His Cys Arg Ile Gln Ala Lys Pro Ala Glu Met Trp Ala Leu Ser 195 200 205 cag tcg gag caa tcc cag atc acc acg cac cct att ccc gat gat gat 672Gln Ser Glu Gln Ser Gln Ile Thr Thr His Pro Ile Pro Asp Asp Asp 210 215 220 cag ggc ggc gag ctc gtc att ggt tcg tcg gca gtg ttc aag gtg ctc 720Gln Gly Gly Glu Leu Val Ile Gly Ser Ser Ala Val Phe Lys Val Leu 225 230 235 240 cgt cac aaa aca gcc tgt gtg gca gag act gat gcg act gtt ctt tta 768Arg His Lys Thr Ala Cys Val Ala Glu Thr Asp Ala Thr Val Leu Leu 245 250 255

ctt gga gaa agt ggg agc ggc aag agc ctt att gca cgc gag atc cac 816Leu Gly Glu Ser Gly Ser Gly Lys Ser Leu Ile Ala Arg Glu Ile His 260 265 270 cgt ttg agc aac aga gcc gac caa gct ttc gtc gaa gtg aac tgt gcc 864Arg Leu Ser Asn Arg Ala Asp Gln Ala Phe Val Glu Val Asn Cys Ala 275 280 285 gct ata ccc gat caa tta atc gag tcg gag ctg ttc ggc gta gag cgc 912Ala Ile Pro Asp Gln Leu Ile Glu Ser Glu Leu Phe Gly Val Glu Arg 290 295 300 gga gcc ttt aca ggt gcg acc gct acg cga gag ggg cgt ttc gag gcg 960Gly Ala Phe Thr Gly Ala Thr Ala Thr Arg Glu Gly Arg Phe Glu Ala 305 310 315 320 gct cat caa ggc act ctc ttc ctt gac gaa att gcc acc ctc agt atg 1008Ala His Gln Gly Thr Leu Phe Leu Asp Glu Ile Ala Thr Leu Ser Met 325 330 335 acc gct caa agt aaa ctt ctc cgt gta ttg cag aat ggc gaa cta gaa 1056Thr Ala Gln Ser Lys Leu Leu Arg Val Leu Gln Asn Gly Glu Leu Glu 340 345 350 cgt ctc ggt agc aac cgg aca atc cac acc agt gtc cga ctg att gcc 1104Arg Leu Gly Ser Asn Arg Thr Ile His Thr Ser Val Arg Leu Ile Ala 355 360 365 gcc acg aac gct gat ctg aaa aaa gct gtc caa gat ggt cac ttc cga 1152Ala Thr Asn Ala Asp Leu Lys Lys Ala Val Gln Asp Gly His Phe Arg 370 375 380 gag gat ctt tac tat cga ctg aac gtc ttt ccc atc cag att ccc cct 1200Glu Asp Leu Tyr Tyr Arg Leu Asn Val Phe Pro Ile Gln Ile Pro Pro 385 390 395 400 ctg cga gaa cgc cgc gat gat att tca ctc att acc agc gta ctc atc 1248Leu Arg Glu Arg Arg Asp Asp Ile Ser Leu Ile Thr Ser Val Leu Ile 405 410 415 gct cga ttt tct aaa cgc cac gga cgt aag tta aaa ggc ata tcg tct 1296Ala Arg Phe Ser Lys Arg His Gly Arg Lys Leu Lys Gly Ile Ser Ser 420 425 430 gcc gcc atg cag gta ctt atc tac cat gac tgg cca ggc aat att cga 1344Ala Ala Met Gln Val Leu Ile Tyr His Asp Trp Pro Gly Asn Ile Arg 435 440 445 gaa ttg gag aac gtg att gag aga gcc ata atc atg gct caa gac gtc 1392Glu Leu Glu Asn Val Ile Glu Arg Ala Ile Ile Met Ala Gln Asp Val 450 455 460 gat ttt ttg gac acc cac cat tta acg aca att gaa ggc acc ctc act 1440Asp Phe Leu Asp Thr His His Leu Thr Thr Ile Glu Gly Thr Leu Thr 465 470 475 480 aca caa gac ttc cta agt ctt aac cag aaa ggt gat cta acc ttg agc 1488Thr Gln Asp Phe Leu Ser Leu Asn Gln Lys Gly Asp Leu Thr Leu Ser 485 490 495 agc gag cta ata cgg aac gcg gct gag aac gcc aac cct aaa gtt ctc 1536Ser Glu Leu Ile Arg Asn Ala Ala Glu Asn Ala Asn Pro Lys Val Leu 500 505 510 tca tta gat gag ttt gcc gag cag atg gtg cat caa ggg tct att aac 1584Ser Leu Asp Glu Phe Ala Glu Gln Met Val His Gln Gly Ser Ile Asn 515 520 525 ctg gac cag gtt caa gac gcc atc acc cga gca gct gtc aaa cat agc 1632Leu Asp Gln Val Gln Asp Ala Ile Thr Arg Ala Ala Val Lys His Ser 530 535 540 ggt ggc aat att tcc cgg gcg gcc tcc ctt ctt ggg ata act cga gcc 1680Gly Gly Asn Ile Ser Arg Ala Ala Ser Leu Leu Gly Ile Thr Arg Ala 545 550 555 560 cgg ctt gat tac cgc gtc aaa aag atc aca tag 1713Arg Leu Asp Tyr Arg Val Lys Lys Ile Thr 565 570 112570PRTEscherichia coli 112Met Lys Ser Asn Lys Asn Asn Ser Asp Asp Arg Ser Ile Val Ala Asp 1 5 10 15 Leu Ala Leu Pro Glu Val His Ala Leu Val Ser Lys Leu His Phe Ser 20 25 30 Pro Asn Glu Gly Arg Ile Trp Leu Asp Glu Ser Arg Cys Leu Leu Leu 35 40 45 Gln Val Glu Thr Leu Lys Asp Ile Tyr Lys Glu Leu Gln Ala Tyr Ser 50 55 60 Gly Pro Asp Tyr Thr Arg Glu Phe Leu Thr Arg Ile Gly Phe Thr Thr 65 70 75 80 Gly Gln Arg Asp Ala Glu Met Ile Ile Lys Lys Gln Gly Ile Ser Ser 85 90 95 Ile Lys Glu Gln Ile Tyr Ala Gly Gly Val Leu His Ala Leu Gln Gly 100 105 110 Phe Leu Thr Ser Ile Gln Ala Gly Ser Ser Ala Leu Asn Ala Val Asp 115 120 125 Met Lys Ser Met Asp Tyr His Ala Glu Ala Tyr Trp Gln Asn Ser Ile 130 135 140 Glu Ala Glu Ile His Leu Ala Met His Gly Val Ser Ser His Ala Val 145 150 155 160 Cys Trp Phe Ser Val Ala Tyr Cys Ser Gly Tyr Leu Ser Ala Cys Ala 165 170 175 Gly Lys Pro Ile Val Val Glu Glu Ile Glu Cys Gln Ala Met Gly His 180 185 190 Thr His Cys Arg Ile Gln Ala Lys Pro Ala Glu Met Trp Ala Leu Ser 195 200 205 Gln Ser Glu Gln Ser Gln Ile Thr Thr His Pro Ile Pro Asp Asp Asp 210 215 220 Gln Gly Gly Glu Leu Val Ile Gly Ser Ser Ala Val Phe Lys Val Leu 225 230 235 240 Arg His Lys Thr Ala Cys Val Ala Glu Thr Asp Ala Thr Val Leu Leu 245 250 255 Leu Gly Glu Ser Gly Ser Gly Lys Ser Leu Ile Ala Arg Glu Ile His 260 265 270 Arg Leu Ser Asn Arg Ala Asp Gln Ala Phe Val Glu Val Asn Cys Ala 275 280 285 Ala Ile Pro Asp Gln Leu Ile Glu Ser Glu Leu Phe Gly Val Glu Arg 290 295 300 Gly Ala Phe Thr Gly Ala Thr Ala Thr Arg Glu Gly Arg Phe Glu Ala 305 310 315 320 Ala His Gln Gly Thr Leu Phe Leu Asp Glu Ile Ala Thr Leu Ser Met 325 330 335 Thr Ala Gln Ser Lys Leu Leu Arg Val Leu Gln Asn Gly Glu Leu Glu 340 345 350 Arg Leu Gly Ser Asn Arg Thr Ile His Thr Ser Val Arg Leu Ile Ala 355 360 365 Ala Thr Asn Ala Asp Leu Lys Lys Ala Val Gln Asp Gly His Phe Arg 370 375 380 Glu Asp Leu Tyr Tyr Arg Leu Asn Val Phe Pro Ile Gln Ile Pro Pro 385 390 395 400 Leu Arg Glu Arg Arg Asp Asp Ile Ser Leu Ile Thr Ser Val Leu Ile 405 410 415 Ala Arg Phe Ser Lys Arg His Gly Arg Lys Leu Lys Gly Ile Ser Ser 420 425 430 Ala Ala Met Gln Val Leu Ile Tyr His Asp Trp Pro Gly Asn Ile Arg 435 440 445 Glu Leu Glu Asn Val Ile Glu Arg Ala Ile Ile Met Ala Gln Asp Val 450 455 460 Asp Phe Leu Asp Thr His His Leu Thr Thr Ile Glu Gly Thr Leu Thr 465 470 475 480 Thr Gln Asp Phe Leu Ser Leu Asn Gln Lys Gly Asp Leu Thr Leu Ser 485 490 495 Ser Glu Leu Ile Arg Asn Ala Ala Glu Asn Ala Asn Pro Lys Val Leu 500 505 510 Ser Leu Asp Glu Phe Ala Glu Gln Met Val His Gln Gly Ser Ile Asn 515 520 525 Leu Asp Gln Val Gln Asp Ala Ile Thr Arg Ala Ala Val Lys His Ser 530 535 540 Gly Gly Asn Ile Ser Arg Ala Ala Ser Leu Leu Gly Ile Thr Arg Ala 545 550 555 560 Arg Leu Asp Tyr Arg Val Lys Lys Ile Thr 565 570 1131584DNAEscherichia coliCDS(1)..(1584)CapR gene encoding CapR biosensor 113atg ctg ttg ctg caa gtt tca gca atg gcc agc ttt cgc cgg gaa atg 48Met Leu Leu Leu Gln Val Ser Ala Met Ala Ser Phe Arg Arg Glu Met 1 5 10 15 gtc aat acc ctg ggc atc gaa cgc gcc aag ggc ttc ttc ctg cgc cag 96Val Asn Thr Leu Gly Ile Glu Arg Ala Lys Gly Phe Phe Leu Arg Gln 20 25 30 ggt tac cag tcc ggc ctg aag gat gcc gaa ctg gcc agg aag ctt aga 144Gly Tyr Gln Ser Gly Leu Lys Asp Ala Glu Leu Ala Arg Lys Leu Arg 35 40 45 ccg aat gcc agc gag tac gac atg ttc ctc gcc ggc ccg cag ctg cat 192Pro Asn Ala Ser Glu Tyr Asp Met Phe Leu Ala Gly Pro Gln Leu His 50 55 60 tcg ctc aag ggt ctg gtc aag gtc cgc ccc acc gag gtc gat atc gac 240Ser Leu Lys Gly Leu Val Lys Val Arg Pro Thr Glu Val Asp Ile Asp 65 70 75 80 aag gaa tgc ggg cgc ttc tat gcc gag atg gag tgg atc gac tcc ttc 288Lys Glu Cys Gly Arg Phe Tyr Ala Glu Met Glu Trp Ile Asp Ser Phe 85 90 95 gag gtg gaa atc tgc cag acc gac ctg ggg cag atg caa gac ccg gtg 336Glu Val Glu Ile Cys Gln Thr Asp Leu Gly Gln Met Gln Asp Pro Val 100 105 110 tgc tgg act ctg ctc ggc tac gcc tgc gcc tat tcc tcg gcg ttc atg 384Cys Trp Thr Leu Leu Gly Tyr Ala Cys Ala Tyr Ser Ser Ala Phe Met 115 120 125 ggc cgg gaa atc atc ttc aag gaa gta agc tgc cgc ggc tgc ggc ggc 432Gly Arg Glu Ile Ile Phe Lys Glu Val Ser Cys Arg Gly Cys Gly Gly 130 135 140 gac aag tgc cgg gtc att ggc aag ccg gcc gaa gag tgg gac gac gtt 480Asp Lys Cys Arg Val Ile Gly Lys Pro Ala Glu Glu Trp Asp Asp Val 145 150 155 160 gcc agc ttc aaa cag tat ttc aag aac gac ccc atc atc gag gaa ctc 528Ala Ser Phe Lys Gln Tyr Phe Lys Asn Asp Pro Ile Ile Glu Glu Leu 165 170 175 tac gag ttg caa tcg caa ctg gtg tcg ctg cgt acc aac ctc gac aaa 576Tyr Glu Leu Gln Ser Gln Leu Val Ser Leu Arg Thr Asn Leu Asp Lys 180 185 190 cag gaa ggc cag tac tac ggc atc ggt cag acc ccg gcc tac cag acc 624Gln Glu Gly Gln Tyr Tyr Gly Ile Gly Gln Thr Pro Ala Tyr Gln Thr 195 200 205 gtg cgc aat atg atg gac aag gcc gca cag ggc aaa gtc tcg gtg ctg 672Val Arg Asn Met Met Asp Lys Ala Ala Gln Gly Lys Val Ser Val Leu 210 215 220 ctg ctt ggc gag acc ggg gtc ggc aag gag gtc atc gcg cgt agc gtg 720Leu Leu Gly Glu Thr Gly Val Gly Lys Glu Val Ile Ala Arg Ser Val 225 230 235 240 cac ctg cgc agc aaa cgc gcc gcc gag ccc ttt gtc gcg gtg aac tgt 768His Leu Arg Ser Lys Arg Ala Ala Glu Pro Phe Val Ala Val Asn Cys 245 250 255 gcg gcg atc ccg ccg gac ctg atc gag tcc gaa ttg ttc ggc gtg gaa 816Ala Ala Ile Pro Pro Asp Leu Ile Glu Ser Glu Leu Phe Gly Val Glu 260 265 270 aaa ggc gcc ttc acc ggc gcc acc cag tca cgc atg ggc cgc ttc gag 864Lys Gly Ala Phe Thr Gly Ala Thr Gln Ser Arg Met Gly Arg Phe Glu 275 280 285 cgg gcc gac aag ggc acc atc ttc ctt gac gag gtg atc gaa ctc agc 912Arg Ala Asp Lys Gly Thr Ile Phe Leu Asp Glu Val Ile Glu Leu Ser 290 295 300 ccg cgc gct cag gcc agt ctg ctg cgc gtg ctg caa gaa ggc gag ctg 960Pro Arg Ala Gln Ala Ser Leu Leu Arg Val Leu Gln Glu Gly Glu Leu 305 310 315 320 gag cga gtt ggc gac aac cgc acg cgc aag atc gac gta agg gtt atc 1008Glu Arg Val Gly Asp Asn Arg Thr Arg Lys Ile Asp Val Arg Val Ile 325 330 335 gca gcc acc cac gag gac ctg gcc gaa gcg gtc aag gcc ggg cgt ttt 1056Ala Ala Thr His Glu Asp Leu Ala Glu Ala Val Lys Ala Gly Arg Phe 340 345 350 cgc gcc gac ctg tac tac cgg ctg aac gtt ttc ccg gtg gcg atc ccg 1104Arg Ala Asp Leu Tyr Tyr Arg Leu Asn Val Phe Pro Val Ala Ile Pro 355 360 365 gcg ttg cgc gaa cgc cgc gag gac att cca ctg ctg gtt gag cac ttc 1152Ala Leu Arg Glu Arg Arg Glu Asp Ile Pro Leu Leu Val Glu His Phe 370 375 380 ctt cag cgc ttc cac cag gag tac ggc aag aga acc ctc ggc ctt tca 1200Leu Gln Arg Phe His Gln Glu Tyr Gly Lys Arg Thr Leu Gly Leu Ser 385 390 395 400 gac aaa gcc ctg gag gcc tgc ctg cat tac agt tgg ccg ggc aat atc 1248Asp Lys Ala Leu Glu Ala Cys Leu His Tyr Ser Trp Pro Gly Asn Ile 405 410 415 cgt gag ctg gag aac gtc atc gag cgc ggc atc atc ctc acc gat ccg 1296Arg Glu Leu Glu Asn Val Ile Glu Arg Gly Ile Ile Leu Thr Asp Pro 420 425 430 aac gaa agc atc agc gtg cag gcg ctg ttc cca cgg gcg ccg gaa gag 1344Asn Glu Ser Ile Ser Val Gln Ala Leu Phe Pro Arg Ala Pro Glu Glu 435 440 445 ccg cag acc gcc agc gag cgg gtg tcg tcg gac ggc gtg ctg att cag 1392Pro Gln Thr Ala Ser Glu Arg Val Ser Ser Asp Gly Val Leu Ile Gln 450 455 460 cca ggc aat ggc cag ggc agt tgg atc agc cag ttg ttg agc agc ggc 1440Pro Gly Asn Gly Gln Gly Ser Trp Ile Ser Gln Leu Leu Ser Ser Gly 465 470 475 480 ctg agc ctc gac gag atc gag gaa agc ctg atg cgc gaa gcc atg caa 1488Leu Ser Leu Asp Glu Ile Glu Glu Ser Leu Met Arg Glu Ala Met Gln 485 490 495 cag gcc aac caa aac gtc tcc ggt gcc gcg cgc ttg ctc ggc cta agc 1536Gln Ala Asn Gln Asn Val Ser Gly Ala Ala Arg Leu Leu Gly Leu Ser 500 505 510 cga ccg gca ctg gcc tat cgg ctg aag aaa atc ggc atc gaa ggc tag 1584Arg Pro Ala Leu Ala Tyr Arg Leu Lys Lys Ile Gly Ile Glu Gly 515 520 525 114527PRTEscherichia coli 114Met Leu Leu Leu Gln Val Ser Ala Met Ala Ser Phe Arg Arg Glu Met 1 5 10 15 Val Asn Thr Leu Gly Ile Glu Arg Ala Lys Gly Phe Phe Leu Arg Gln 20 25 30 Gly Tyr Gln Ser Gly Leu Lys Asp Ala Glu Leu Ala Arg Lys Leu Arg 35 40 45 Pro Asn Ala Ser Glu Tyr Asp Met Phe Leu Ala Gly Pro Gln Leu His 50 55 60 Ser Leu Lys Gly Leu Val Lys Val Arg Pro Thr Glu Val Asp Ile Asp 65 70 75 80 Lys Glu Cys Gly Arg Phe Tyr Ala Glu Met Glu Trp Ile Asp Ser Phe 85 90 95 Glu Val Glu Ile Cys Gln Thr Asp Leu Gly Gln Met Gln Asp Pro Val 100 105 110 Cys Trp Thr Leu Leu Gly Tyr Ala Cys Ala Tyr Ser Ser Ala Phe Met 115 120 125 Gly Arg Glu Ile Ile Phe Lys Glu Val Ser Cys Arg Gly Cys Gly Gly 130 135 140 Asp Lys Cys Arg Val Ile Gly Lys Pro Ala Glu Glu Trp Asp Asp Val 145 150 155 160 Ala Ser Phe Lys Gln Tyr Phe Lys Asn Asp Pro Ile Ile Glu Glu Leu 165 170 175 Tyr Glu Leu Gln Ser Gln Leu Val Ser Leu Arg Thr Asn Leu Asp Lys 180 185 190 Gln Glu Gly Gln Tyr Tyr Gly Ile Gly Gln Thr Pro Ala Tyr Gln Thr 195 200 205 Val Arg Asn Met Met Asp Lys Ala Ala Gln Gly Lys Val Ser Val Leu 210 215 220 Leu Leu Gly Glu Thr Gly Val Gly Lys Glu Val Ile Ala Arg Ser Val 225 230 235 240 His Leu Arg Ser Lys Arg Ala Ala Glu Pro Phe Val Ala Val Asn Cys 245 250 255 Ala Ala Ile Pro Pro Asp Leu Ile Glu Ser Glu Leu Phe Gly Val

Glu 260 265 270 Lys Gly Ala Phe Thr Gly Ala Thr Gln Ser Arg Met Gly Arg Phe Glu 275 280 285 Arg Ala Asp Lys Gly Thr Ile Phe Leu Asp Glu Val Ile Glu Leu Ser 290 295 300 Pro Arg Ala Gln Ala Ser Leu Leu Arg Val Leu Gln Glu Gly Glu Leu 305 310 315 320 Glu Arg Val Gly Asp Asn Arg Thr Arg Lys Ile Asp Val Arg Val Ile 325 330 335 Ala Ala Thr His Glu Asp Leu Ala Glu Ala Val Lys Ala Gly Arg Phe 340 345 350 Arg Ala Asp Leu Tyr Tyr Arg Leu Asn Val Phe Pro Val Ala Ile Pro 355 360 365 Ala Leu Arg Glu Arg Arg Glu Asp Ile Pro Leu Leu Val Glu His Phe 370 375 380 Leu Gln Arg Phe His Gln Glu Tyr Gly Lys Arg Thr Leu Gly Leu Ser 385 390 395 400 Asp Lys Ala Leu Glu Ala Cys Leu His Tyr Ser Trp Pro Gly Asn Ile 405 410 415 Arg Glu Leu Glu Asn Val Ile Glu Arg Gly Ile Ile Leu Thr Asp Pro 420 425 430 Asn Glu Ser Ile Ser Val Gln Ala Leu Phe Pro Arg Ala Pro Glu Glu 435 440 445 Pro Gln Thr Ala Ser Glu Arg Val Ser Ser Asp Gly Val Leu Ile Gln 450 455 460 Pro Gly Asn Gly Gln Gly Ser Trp Ile Ser Gln Leu Leu Ser Ser Gly 465 470 475 480 Leu Ser Leu Asp Glu Ile Glu Glu Ser Leu Met Arg Glu Ala Met Gln 485 490 495 Gln Ala Asn Gln Asn Val Ser Gly Ala Ala Arg Leu Leu Gly Leu Ser 500 505 510 Arg Pro Ala Leu Ala Tyr Arg Leu Lys Lys Ile Gly Ile Glu Gly 515 520 525 115434DNAEscherichia colipromoter(1)..(434)gudP promoter 115tatgttcagc gagcggtaaa tgtcgtttta gcggtgctga atcgaatctt ttcaggcaaa 60tgccagtaaa aactgcttca tagcgcggat ttttactggc gtttgcctgg agtcaagcga 120tccatttcat actcttcttt atttcttcgt tttaaccctt cctttcttgt tcttgttttc 180atttccgtga agtggattcc accgtccagg gctaatgcca aaatcgggcc tcattgaacg 240cattaatgtt gtgttgttgc acggtgagcc gctatggcgc gctttttata ctgctattgc 300cagatataaa cacgcgccgt attcggcgaa cgacctataa aaacggcaaa aaacacccta 360cgtcacctct gatttcctgg cgatgtcgca gtccagagtg agcgtggcta acgcgaattt 420tcaggagtgc aaca 4341161158DNAEscherichia coliCDS(1)..(1158)CdaR gene encoding CdaR biosensor 116atg gct ggc tgg cat ctt gat acc aaa atg gcg cag gat atc gtg gca 48Met Ala Gly Trp His Leu Asp Thr Lys Met Ala Gln Asp Ile Val Ala 1 5 10 15 cgt acc atg cgc atc atc gat acc aat atc aac gta atg gat gcc cgt 96Arg Thr Met Arg Ile Ile Asp Thr Asn Ile Asn Val Met Asp Ala Arg 20 25 30 ggg cga att atc ggc agc ggc gat cgt gag cgt att ggt gaa ttg cac 144Gly Arg Ile Ile Gly Ser Gly Asp Arg Glu Arg Ile Gly Glu Leu His 35 40 45 gaa ggt gca ttg ctg gta ctt tca cag gga cga gtc gtc gat atc gat 192Glu Gly Ala Leu Leu Val Leu Ser Gln Gly Arg Val Val Asp Ile Asp 50 55 60 gac gcg gta gca cgt cat ctg cac ggt gtg cgg cag ggg att aat cta 240Asp Ala Val Ala Arg His Leu His Gly Val Arg Gln Gly Ile Asn Leu 65 70 75 80 ccg tta cgg ctg gaa ggt gaa att gtc ggc gta att ggc ctg aca ggt 288Pro Leu Arg Leu Glu Gly Glu Ile Val Gly Val Ile Gly Leu Thr Gly 85 90 95 gaa cca gag aat ctg cgt aaa tat ggc gaa ctg gtc tgc atg acg gct 336Glu Pro Glu Asn Leu Arg Lys Tyr Gly Glu Leu Val Cys Met Thr Ala 100 105 110 gaa atg atg ctg gaa cag tcg cgg ttg atg cac ttg ttg gcg cag gat 384Glu Met Met Leu Glu Gln Ser Arg Leu Met His Leu Leu Ala Gln Asp 115 120 125 agc cgt ttg cgg gaa gaa ctg gtg atg aac ctg att cag gca gag gag 432Ser Arg Leu Arg Glu Glu Leu Val Met Asn Leu Ile Gln Ala Glu Glu 130 135 140 aat act ccc gca ctt act gaa tgg gcg caa cgg ctg ggg atc gat ctc 480Asn Thr Pro Ala Leu Thr Glu Trp Ala Gln Arg Leu Gly Ile Asp Leu 145 150 155 160 aat caa ccg cga gtg gtg gct att gtt gag gtc gac agc ggt cag ctt 528Asn Gln Pro Arg Val Val Ala Ile Val Glu Val Asp Ser Gly Gln Leu 165 170 175 ggc gtg gac agc gca atg gcg gag tta caa caa ctg caa aac gcg ctg 576Gly Val Asp Ser Ala Met Ala Glu Leu Gln Gln Leu Gln Asn Ala Leu 180 185 190 act acg ccc gag cgt aat aat ctg gtg gcg att gtc tcg cta acc gaa 624Thr Thr Pro Glu Arg Asn Asn Leu Val Ala Ile Val Ser Leu Thr Glu 195 200 205 atg gtg gtg ttg aaa ccg gcg ttg aac tct ttt ggg cgc tgg gat gca 672Met Val Val Leu Lys Pro Ala Leu Asn Ser Phe Gly Arg Trp Asp Ala 210 215 220 gaa gat cat cgt aag cga gtt gaa caa ctg att acc cgc atg aaa gag 720Glu Asp His Arg Lys Arg Val Glu Gln Leu Ile Thr Arg Met Lys Glu 225 230 235 240 tac ggc cag ctg cgt ttt cgc gtt tca ctg ggc aac tat ttt acc ggt 768Tyr Gly Gln Leu Arg Phe Arg Val Ser Leu Gly Asn Tyr Phe Thr Gly 245 250 255 cct ggc agt att gcc cga tcc tat cgt acg gcg aaa acg acg atg gtg 816Pro Gly Ser Ile Ala Arg Ser Tyr Arg Thr Ala Lys Thr Thr Met Val 260 265 270 gtg ggt aaa cag cgg atg cca gaa agt cgc tgc tat ttt tat cag gat 864Val Gly Lys Gln Arg Met Pro Glu Ser Arg Cys Tyr Phe Tyr Gln Asp 275 280 285 ctg atg tta cct gtg tta ctc gac agt ttg cgt ggc gac tgg cag gcc 912Leu Met Leu Pro Val Leu Leu Asp Ser Leu Arg Gly Asp Trp Gln Ala 290 295 300 aac gaa ctg gcg cga ccg ctg gcg cgg ctg aaa acg atg gac aat aac 960Asn Glu Leu Ala Arg Pro Leu Ala Arg Leu Lys Thr Met Asp Asn Asn 305 310 315 320 ggc ttg ctg cga cga acg ctg gcg gcg tgg ttt cgc cac aat gtg caa 1008Gly Leu Leu Arg Arg Thr Leu Ala Ala Trp Phe Arg His Asn Val Gln 325 330 335 ccg ctg gca acg tca aag gcg ttg ttt att cat cgt aat acc ctg gag 1056Pro Leu Ala Thr Ser Lys Ala Leu Phe Ile His Arg Asn Thr Leu Glu 340 345 350 tat cgg ctt aat cgt ata tcg gaa ctg acc ggg ctt gat ttg ggc aat 1104Tyr Arg Leu Asn Arg Ile Ser Glu Leu Thr Gly Leu Asp Leu Gly Asn 355 360 365 ttt gat gac agg ttg ctg ctg tat gtg gcg tta caa ctg gat gaa gag 1152Phe Asp Asp Arg Leu Leu Leu Tyr Val Ala Leu Gln Leu Asp Glu Glu 370 375 380 cgg tag 1158Arg 385 117385PRTEscherichia coli 117Met Ala Gly Trp His Leu Asp Thr Lys Met Ala Gln Asp Ile Val Ala 1 5 10 15 Arg Thr Met Arg Ile Ile Asp Thr Asn Ile Asn Val Met Asp Ala Arg 20 25 30 Gly Arg Ile Ile Gly Ser Gly Asp Arg Glu Arg Ile Gly Glu Leu His 35 40 45 Glu Gly Ala Leu Leu Val Leu Ser Gln Gly Arg Val Val Asp Ile Asp 50 55 60 Asp Ala Val Ala Arg His Leu His Gly Val Arg Gln Gly Ile Asn Leu 65 70 75 80 Pro Leu Arg Leu Glu Gly Glu Ile Val Gly Val Ile Gly Leu Thr Gly 85 90 95 Glu Pro Glu Asn Leu Arg Lys Tyr Gly Glu Leu Val Cys Met Thr Ala 100 105 110 Glu Met Met Leu Glu Gln Ser Arg Leu Met His Leu Leu Ala Gln Asp 115 120 125 Ser Arg Leu Arg Glu Glu Leu Val Met Asn Leu Ile Gln Ala Glu Glu 130 135 140 Asn Thr Pro Ala Leu Thr Glu Trp Ala Gln Arg Leu Gly Ile Asp Leu 145 150 155 160 Asn Gln Pro Arg Val Val Ala Ile Val Glu Val Asp Ser Gly Gln Leu 165 170 175 Gly Val Asp Ser Ala Met Ala Glu Leu Gln Gln Leu Gln Asn Ala Leu 180 185 190 Thr Thr Pro Glu Arg Asn Asn Leu Val Ala Ile Val Ser Leu Thr Glu 195 200 205 Met Val Val Leu Lys Pro Ala Leu Asn Ser Phe Gly Arg Trp Asp Ala 210 215 220 Glu Asp His Arg Lys Arg Val Glu Gln Leu Ile Thr Arg Met Lys Glu 225 230 235 240 Tyr Gly Gln Leu Arg Phe Arg Val Ser Leu Gly Asn Tyr Phe Thr Gly 245 250 255 Pro Gly Ser Ile Ala Arg Ser Tyr Arg Thr Ala Lys Thr Thr Met Val 260 265 270 Val Gly Lys Gln Arg Met Pro Glu Ser Arg Cys Tyr Phe Tyr Gln Asp 275 280 285 Leu Met Leu Pro Val Leu Leu Asp Ser Leu Arg Gly Asp Trp Gln Ala 290 295 300 Asn Glu Leu Ala Arg Pro Leu Ala Arg Leu Lys Thr Met Asp Asn Asn 305 310 315 320 Gly Leu Leu Arg Arg Thr Leu Ala Ala Trp Phe Arg His Asn Val Gln 325 330 335 Pro Leu Ala Thr Ser Lys Ala Leu Phe Ile His Arg Asn Thr Leu Glu 340 345 350 Tyr Arg Leu Asn Arg Ile Ser Glu Leu Thr Gly Leu Asp Leu Gly Asn 355 360 365 Phe Asp Asp Arg Leu Leu Leu Tyr Val Ala Leu Gln Leu Asp Glu Glu 370 375 380 Arg 385 118136DNAEscherichia colipromoter(1)..(136)pSAL promoter 118ggggcctcgc ttgggttatt gctggtgccc ggccgggcgc aatattcatg ttgatgattt 60attatatatc gagtggtgta tttatcaata ttgtttgctc cgttatcgtt attaacaagt 120catcaataaa gccatc 136

Claims

1. A genetically modified microbial cell for intracellular biosynthesis of a cellular metabolite comprising: a. a first nucleic acid molecule wherein the transcription and/or translation of said molecule yields a biosensor capable of binding said cellular metabolite to form a complex; and any one selected from the group consisting of: b. a second nucleic acid molecule comprising a coding sequence encoding a first protein required for cell growth and/or survival, wherein the second nucleic acid molecule is operably linked to a first promoter; wherein expression of said first protein encoded by said second nucleic acid molecule is induced when said biosensor and said cellular metabolite form a complex; c. a second nucleic acid molecule comprising a coding sequence encoding a first protein required for cell growth and/or survival, wherein the second nucleic acid molecule is operably linked to a first promoter; and a third nucleic acid molecule encoding a second protein that is toxic for cell growth and/or survival, wherein said third nucleic acid molecule comprises a coding sequence operably linked to a second constitutive promoter; wherein expression of said first protein encoded by said second nucleic acid molecule is induced when said biosensor and said cellular metabolite form a complex; and d. a second nucleic acid molecule encoding a protein that is toxic for cell growth and/or survival, wherein said second nucleic acid molecule comprises a coding sequence operably linked to a promoter; wherein expression of said protein is prevented when said biosensor and said cellular metabolite form a complex; whereby arrest of growth and/or death of said cell due to an absence of complex formation does not depend on externally supplied growth inhibitor or growth retardant.

2. The genetically modified microbial cell of claim 1 according to (b), wherein said first promoter is an inducible promoter, and said encoded first protein is essential for growth of the cell.

3. The genetically modified microbial cell of claim 1 according to (c), wherein: said second protein is a toxin; and said first protein is an anti-toxin protein cognate to said toxin protein; and said first promoter is inducible, and wherein said biosensor is a transcription factor capable of binding to said metabolite to form a complex, and wherein said complex is capable of binding to said first inducible promoter.

4. The genetically modified microbial cell of claim 1 according to (c), wherein: said second protein is a toxin; and said first protein is an anti-toxin protein cognate to said toxin protein; and said first nucleic acid molecule is operably linked to said second nucleic acid molecule upstream to the coding sequence and is operably linked downstream of the first promoter, wherein the first promoter is a constitutive promoter, and wherein said biosensor obtained on transcription of said first nucleic acid molecule is a riboswitch capable of binding to said metabolite to form a complex.

5. The genetically modified microbial cell of claim 1 according to (d), wherein: said protein is a toxin; and said promoter is inducible; and wherein said biosensor is a transcription factor capable of binding to said metabolite to form a complex, and wherein said complex is capable of binding to said inducible promoter.

6. The genetically modified microbial cell of claim 1 according to (d), wherein: said protein is a toxin; said first nucleic acid molecule is operably linked to said second nucleic acid molecule upstream to the coding sequence and is operably linked downstream of the first promoter, and wherein the first promoter is a constitutive promoter, and wherein said biosensor obtained on transcription of said first nucleic acid molecule is a riboswitch capable of binding to said metabolite to form a complex.

7. The genetically modified microbial cell of claim 3, wherein the amino acid sequence of said antitoxin has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID No.: 2, 38, 42, and 46; and wherein the amino acid sequence of said cognate toxin protein has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID No.: 3, 36, 40 and 44 respectively.

8. The genetically modified microbial cell of claim 5, wherein the amino acid sequence of said toxin protein has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID No.: 3, 36, 40 and 44 respectively.

9. The genetically modified microbial cell of claim 1, wherein said biosensor obtained on transcription of said first nucleic acid molecule is a riboswitch capable of binding to said metabolite to form a complex, and wherein the nucleic acid sequence of said a first nucleic acid molecule is selected from the group consisting of: nucleotides sequence 1291-1452 of SEQ ID No.: 31; SEQ ID No.:69, 70 and 71.

10. The genetically modified microbial cell of claim 1, wherein said biosensor obtained on translation of said first nucleic acid molecule is a transcription factor capable of binding to said metabolite to form a complex, and wherein the nucleic acid sequence of said a first nucleic acid molecule is selected from the group consisting of: SEQ ID No.:6, 12 and 68.

11. The genetically modified microbial cell of claim 2, wherein the amino acid sequence of said essential protein has at least 80% sequence identity to a sequence selected for the group consisting of: SEQ ID No.: 76, 78, 80, 82, 84, 86 and 88.

12. The genetically modified microbial cell of claim 1, wherein the cellular metabolite is selected from the group consisting of: isoprenoid(s), vitamin(s), carboxylic acid(s), amino acid(s), fatty acid(s), alcohol(s), and polyketide(s).

13. A method of genetically modifying a microbial cell for the biosynthesis of a metabolite comprising the steps of introducing into the cell: a nucleic acid molecule encoding a toxin operably linked to a constitutive promoter; and a nucleic acid molecule encoding an anti-toxin cognate to the toxin, wherein the molecule is linked to a inducible promoter; and a nucleic acid molecule wherein the transcription and/or translation of said molecule yields a biosensor capable of binding to the metabolite; wherein expression of said antitoxin is induced when said biosensor and said cellular metabolite form a complex; and wherein arrest of growth and/or death of said cell due to an absence of complex formation does not depend on externally supplied growth inhibitor or growth retardant.

14. A method for producing a biosynthetic metabolite comprising the steps of: a) providing a genetically modified microbial cell according to claim 1, b) introducing the genetically modified microbial cell into a cultivation medium comprising a substrate for production of said metabolite, and c) recovering metabolite produced by said culture, wherein a lack of metabolite production in said genetically modified microbial cell or progeny cell thereof attenuates multiplication of said cell as compared to a non-genetically modified parent cell from which said modified microbial cell was derived.

15. A biosynthetic metabolite produced by the genetically modified microbial cell according to claim 1, wherein a lack of metabolite production in said genetically modified microbial cell or progeny cell thereof attenuates multiplication of said cell as compared to the metabolite producing genetically modified microbial cell.

16. The genetically modified microbial cell of claim 4, wherein the amino acid sequence of said antitoxin has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID No.: 2, 38, 42, and 46; and wherein the amino acid sequence of said cognate toxin protein has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID No.: 3, 36, 40 and 44 respectively.

17. The genetically modified microbial cell of claim 6, wherein the amino acid sequence of said toxin protein has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID No.: 3, 36, 40 and 44 respectively.